Protein-protein interactions

ABSTRACT

The present invention relates to the discovery of novel protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases. Examples of physiological disorders and diseases include non-insulin dependent diabetes mellitus (NIDDM), neurodegenerative disorders, such as Alzheimer&#39;s Disease (AD), and the like. Thus, the present invention is directed to complexes of these proteins and/or their fragments, antibodies to the complexes, diagnosis of physiological generative disorders (including diagnosis of a predisposition to and diagnosis of the existence of the disorder), drug screening for agents which modulate the interaction of proteins described herein, and identification of additional proteins in the pathway common to the proteins described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is related to U.S. provisional patent applications Serial No. 60/168,377 filed Dec. 2, 1999, Serial No. 60/168,379 filed Dec. 2, 1999 and Serial No. 60/185,056 filed Feb. 25, 2000, each incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the discovery of novel protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases.

[0003] Examples of physiological disorders and diseases include non-insulin dependent diabetes mellitus (NIDDM), neurodegenerative disorders, such as Alzheimer's Disease (AD), and the like. Thus, the present invention is directed to complexes of these proteins and/or their fragments, antibodies to the complexes, diagnosis of physiological generative disorders (including diagnosis of a predisposition to and diagnosis of the existence of the disorder), drug screening for agents which modulate the interaction of proteins described herein, and identification of additional proteins in the pathway common to the proteins described herein.

[0004] The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References.

[0005] Many processes in biology, including transcription, translation and metabolic or signal transduction pathways, are mediated by non-covalently associated protein complexes. The formation of protein-protein complexes or protein-DNA complexes produce the most efficient chemical machinery. Much of modern biological research is concerned with identifying proteins involved in cellular processes, determining their functions, and how, when and where they interact with other proteins involved in specific pathways. Further, with rapid advances in genome sequencing, there is a need to define protein linkage maps, i.e., detailed inventories of protein interactions that make up functional assemblies of proteins or protein complexes or that make up physiological pathways.

[0006] Recent advances in human genomics research has led to rapid progress in the identification of novel genes. In applications to biological and pharmaceutical research, there is a need to determine functions of gene products. A first step in defining the function of a novel gene is to determine its interactions with other gene products in appropriate context. That is, since proteins make specific interactions with other proteins or other biopolymers as part of functional assemblies or physiological pathways, an appropriate way to examine function of a gene is to determine its physical relationship with other genes. Several systems exist for identifying protein interactions and hence relationships between genes.

[0007] There continues to be a need in the art for the discovery of additional protein-protein interactions involved in mammalian physiological pathways. There continues to be a need in the art also to identify the protein-protein interactions that are involved in mammalian physiological disorders and diseases, and to thus identify drug targets.

SUMMARY OF THE INVENTION

[0008] The present invention relates to the discovery of protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases, and to the use of this discovery. The identification of the interacting proteins described herein provide new targets for the identification of useful pharmaceuticals, new targets for diagnostic tools in the identification of individuals at risk, sequences for production of transformed cell lines, cellular models and animal models, and new bases for therapeutic intervention in such physiological pathways Thus, one aspect of the present invention is protein complexes. The protein complexes are a complex of (a) two interacting proteins, (b) a first interacting protein and a fragment of a second interacting protein, (c) a fragment of a first interacting protein and a second interacting protein, or (d) a fragment of a first interacting protein and a fragment of a second interacting protein. The fragments of the interacting proteins include those parts of the proteins, which interact to form a complex. This aspect of the invention includes the detection of protein interactions and the production of proteins by recombinant techniques. The latter embodiment also includes cloned sequences, vectors, transfected or transformed host cells and transgenic animals.

[0009] A second aspect of the present invention is an antibody that is immunoreactive with the above complex. The antibody may be a polyclonal antibody or a monoclonal antibody. While the antibody is immunoreactive with the complex, it is not immunoreactive with the component parts of the complex. That is, the antibody is not immunoreactive with a first interactive protein, a fragment of a first interacting protein, a second interacting protein or a fragment of a second interacting protein. Such antibodies can be used to detect the presence or absence of the protein complexes.

[0010] A third aspect of the present invention is a method for diagnosing a predisposition for physiological disorders or diseases in a human or other animal. The diagnosis of such disorders includes a diagnosis of a predisposition to the disorders and a diagnosis for the existence of the disorders. In accordance with this method, the ability of a first interacting protein or fragment thereof to form a complex with a second interacting protein or a fragment thereof is assayed, or the genes encoding interacting proteins are screened for mutations in interacting portions of the protein molecules. The inability of a first interacting protein or fragment thereof to form a complex, or the presence of mutations in a gene within the interacting domain, is indicative of a predisposition to, or existence of a disorder. In accordance with one embodiment of the invention, the ability to form a complex is assayed in a two-hybrid assay. In a first aspect of this embodiment, the ability to form a complex is assayed by a yeast two-hybrid assay. In a second aspect, the ability to form a complex is assayed by a mammalian two-hybrid assay. In a second embodiment, the ability to form a complex is assayed by measuring in vitro a complex formed by combining said first protein and said second protein. In one aspect the proteins are isolated from a human or other animal. In a third embodiment, the ability to form a complex is assayed by measuring the binding of an antibody, which is specific for the complex. In a fourth embodiment, the ability to form a complex is assayed by measuring the binding of an antibody that is specific for the complex with a tissue extract from a human or other animal. In a fifth embodiment, coding sequences of the interacting proteins described herein are screened for mutations.

[0011] A fourth aspect of the present invention is a method for screening for drug candidates which are capable of modulating the interaction of a first interacting protein and a second interacting protein. In this method, the amount of the complex formed in the presence of a drug is compared with the amount of the complex formed in the absence of the drug. If the amount of complex formed in the presence of the drug is greater than or less than the amount of complex formed in the absence of the drug, the drug is a candidate for modulating the interaction of the first and second interacting proteins. The drug promotes the interaction if the complex formed in the presence of the drug is greater and inhibits (or disrupts) the interaction if the complex formed in the presence of the drug is less. The drug may affect the interaction directly, i.e., by modulating the binding of the two proteins, or indirectly, e.g., by modulating the expression of one or both of the proteins.

[0012] A fifth aspect of the present invention is a model for such physiological pathways, disorders or diseases. The model may be a cellular model or an animal model, as further described herein. In accordance with one embodiment of the invention, an animal model is prepared by creating transgenic or “knock-out” animals. The knock-out may be a total knock-out, i.e., the desired gene is deleted, or a conditional knock-out, i.e., the gene is active until it is knocked out at a determined time. In a second embodiment, a cell line is derived from such animals for use as a model. In a third embodiment, an animal model is prepared in which the biological activity of a protein complex of the present invention has been altered. In one aspect, the biological activity is altered by disrupting the formation of the protein complex, such as by the binding of an antibody or small molecule to one of the proteins which prevents the formation of the protein complex. In a second aspect, the biological activity of a protein complex is altered by disrupting the action of the complex, such as by the binding of an antibody or small molecule to the protein complex which interferes with the action of the protein complex as described herein. In a fourth embodiment, a cell model is prepared by altering the genome of the cells in a cell line. In one aspect, the genome of the cells is modified to produce at least one protein complex described herein. In a second aspect, the genome of the cells is modified to eliminate at least one protein of the protein complexes described herein.

[0013] A sixth aspect of the present invention are nucleic acids coding for novel proteins discovered in accordance with the present invention and the corresponding proteins and antibodies.

[0014] A seventh aspect of the present invention is a method of screening for drug candidates useful for treating a physiological disorder. In this embodiment, drugs are screened on the basis of the association of a protein with a particular physiological disorder. This association is established in accordance with the present invention by identifying a relationship of the protein with a particular physiological disorder. The drugs are screened by comparing the activity of the protein in the presence and absence of the drug. If a difference in activity is found, then the drug is a drug candidate for the physiological disorder. The activity of the protein can be assayed in vitro or in vivo using conventional techniques, including transgenic animals and cell lines of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention is the discovery of novel interactions between proteins described herein. The genes coding for some of these proteins may have been cloned previously, but their potential interaction in a physiological pathway or with a particular protein was unknown. Alternatively, the genes coding for some of these proteins have not been cloned previously and represent novel genes. These proteins are identified using the yeast two-hybrid method and searching a human total brain library, as more filly described below.

[0016] According to the present invention, new protein-protein interactions have been discovered. The discovery of these interactions has identified several protein complexes for each protein-protein interaction. The protein complexes for these interactions are set forth below in Tables 1-31, which also identify the new protein-protein interactions of the present invention. TABLE 1 Protein Complexes of p38 alpha/CYT4 Interaction Protein Kinase p38 alpha (p38 alpha) and CYT4 A fragment of p38 alpha and CYT4 p38 alpha and a fragment of CYT4 A fragment of p38 alpha and a fragment of CYT4

[0017] TABLE 2 Protein Complexes of MAPKAP-K3/PN2012 Interaction MAP Kinase MAPKAP-K3 (MAPKAP-K3) and Novel Protein PN2012 (PN2012) A fragment of MAPKAP-K3 and PN2012 MAPKAP-K3 and a fragment of PN2012 A fragment of MAPKAP-K3 and a fragment of PN2012

[0018] TABLE 3 Protein Complexes of MAPKAP-K3/PN7771 Interaction MAP Kinase MAPKAP-K3 (MAPKAP-K3) and Novel Protein Fragment PN7771 (PN7771) A fragment of MAPKAP-K3 and PN7771 MAPKAP-K3 and a fragment of PN7771 A fragment of MAPKAP-K3 and a fragment of PN7771

[0019] TABLE 4 Protein Complexes of PRAK/PN7098 Interaction Protein Kinase PRAK (PRAK) and Novel Protein Fragment PN7098 (PN7098) A fragment of PRAK and PN7098 PRAK and a fragment of PN7098 A fragment of PRAK and a fragment of PN7098

[0020] TABLE 5 Protein Complexes of PRAK/Kendrin Interaction Protein kinase PRAK (PRAK) and kendrin A fragment of PRAK and kendrin PRAK and a fragment of kendrin A fragment of PRAK and a fragment of kendrin

[0021] TABLE 6 Protein Complexes of PRAK/Homeotic Protein Prox1 Interaction Protein kinase PRAK (PRAK) and Homeotic Protein Prox1 (Prox 1) A fragment of PRAK and Prox1 PRAK and a fragment of Prox1 A fragment of PRAK and a fragment of Prox1

[0022] TABLE 7 Protein Complexes of PRAK/Hook1 Interaction Protein kinase PRAK (PRAK) and Hook1 A fragment of PRAK and Hook1 PRAK and a fragment of Hook1 A fragment of PRAK and a fragment of Hook1

[0023] TABLE 8 Protein Complexes of PRAK/IG Heavy Chain Constant Region Interaction Protein kinase PRAK (PRAK) and IG heavy chain constant region A fragment of PRAK and IG heavy chain constant region PRAK and a fragment of IG heavy chain constant region A fragment of PRAK and a fragment of IG heavy chain constant region

[0024] TABLE 9 Protein Complexes of PRAK/Golgin-95 Interaction Protein kinase PRAK (PRAK) and golgin-95 A fragment of PRAK and golgin-95 PRAK and a fragment of golgin-95 A fragment of PRAK and a fragment of golgin-95

[0025] TABLE 10 Protein Complexes of PRAK/KIAA0555 Interaction Protein kinase PRAK (PRAK) and KIAA0555 A fragment of PRAK and KIAA0555 PRAK and a fragment of KIAA0555 A fragment of PRAK and a fragment of KIAA0555

[0026] TABLE 11 Protein Complexes of PRAK/Leucine-rich Protein L130 Interaction Protein kinase PRAK (PRAK) and leucine-rich protein L130 A fragment of PRAK and leucine-rich protein L130 PRAK and a fragment of leucine-rich protein L130 A fragment of PRAK and a fragment of leucine-rich protein L130

[0027] TABLE 12 Protein Complexes of PRAK/ERK3 Interaction Protein kinase PRAK (PRAK) and ERK3 A fragment of PRAK and ERK3 PRAK and a fragment of ERK3 A fragment of PRAK and a fragment of ERK3

[0028] TABLE 13 Protein Complexes of PRAK/cAMP-dependent Protein Kinase Interaction Protein kinase PRAK (PRAK) and cAMP-dependent protein kinase A fragment of PRAK and cAMP-dependent protein kinase PRAK and a fragment of cAMP-dependent protein kinase A fragment of PRAK and a fragment of cAMP-dependent protein kinase

[0029] TABLE 14 Protein Complexes of PRAK/AL117538 Protein kinase PRAK (PRAK) and AL117538 A fragment of PRAK and AL117538 PRAK and a fragment of AL117538 A fragment of PRAK and a fragment of AL117538

[0030] TABLE 15 Protein Complexes of PRAK/AL117237 Protein kinase PRAK (PRAK) and AL117237 A fragment of PRAK and AL117237 PRAK and a fragment of AL117237 A fragment of PRAK and a fragment of AL117237

[0031] TABLE 16 Protein Complexes of p38 Alpha/JNK3 Alpha2 Interaction Protein Kinase p38 alpha (p38 alpha) and JNK3 alpha2 A fragment of p38 alpha and JNK3 alpha2 p38 alpha and a fragment of JNK3 alpha2 A fragment of p38 alpha and a fragment of JNK3 alpha2

[0032] TABLE 17 Protein Complexes p38 Alpha/C-Nap1 Interaction Protein Kinase p38 alpha (p38 alpha) and C-Nap1 A fragment of p38 alpha and C-Nap1 p38 alpha and a fragment of C-Nap1 A fragment of p38 alpha and a fragment of C-Nap1

[0033] TABLE 18 Protein Complexes p38 Alpha/Vinculin Interaction Protein Kinase p38 alpha (p38 alpha) and Vinculin A fragment of p38 alpha and Vinculin p38 alpha and a fragment of Vinculin A fragment of p38 alpha and a fragment of Vinculin

[0034] TABLE 19 Protein Complexes p38 Alpha K53M Mutant/ Splicing Factor PSF Interaction Protein Kinase p38 alpha (p38 alpha) K53M Mutant and Splicing Factor PSF A fragment of p38 alpha K53M Mutant and Splicing Factor PSF p38 alpha K53M Mutant and a fragment of Splicing Factor PSF A fragment of p38 alpha K53M Mutant and a fragment of Splicing Factor PSF

[0035] TABLE 20 Protein Complexes of MAPKAP-K2/Leucine-rich Protein L130 Interaction MAPKAP-K2 and leucine-rich protein L130 A fragment of MAPKAP-K2 and leucine-rich protein L130 MAPKAP-K2 and a fragment of leucine-rich protein L130 A fragment of MAPKAP-K2 and a fragment of leucine-rich protein L130

[0036] TABLE 21 Protein Complexes of MAPKAP-K2/ cAMP-dependent Protein Kinase Interaction MAPKAP-K2 and cAMP-dependent Protein Kinase A fragment of MAPKAP-K2 and cAMP-dependent Protein Kinase MAPKAP-K2 and a fragment of cAMP-dependent Protein Kinase A fragment of MAPKAP-K2 and a cAMP-dependent Protein Kinase

[0037] TABLE 22 Protein Complexes of MAPKAP-K2/SET Interaction MAPKAP-K2 and SET A fragment of MAPKAP-K2 and SET MAPKAP-K2 and a fragment of SET A fragment of MAPKAP-K2 and a SET

[0038] TABLE 23 Protein Complexes of MAPKAP-K2/TL21 Interaction MAPKAP-K2 and TL21 A fragment of MAPKAP-K2 and TL21 MAPKAP-K2 and a fragment of TL21 A fragment of MAPKAP-K2 and a TL21

[0039] TABLE 24 Protein Complexes of MAPKAP-K2 (K93M, T222D, T334D Mutant)/ERK3 Interaction MAPKAP-K2 K93M, T222D, T334D Mutant and ERK3 A fragment of MAPKAP-K2 K93M, T222D, T334D Mutant and ERK3 MAPKAP-K2 K93M, T222D, T334D Mutant and a fragment of ERK3 A fragment of MAPKAP-K2 K93M, T222D, T334D Mutant and a ERK3

[0040] TABLE 25 Protein Complexes of MAPKAP-K3/Thrombospondin 3 Interaction MAPKAP-K3 and thrombospondin 3 A fragment of MAPKAP-K3 and thrombospondin 3 MAPKAP-K3 and a fragment of thrombospondin 3 A fragment of MAPKAP-K3 and a fragment of thrombospondin 3

[0041] TABLE 26 Protein Complexes of MAPKAP-K3/Malate Dehydrogenase Interaction MAPKAP-K3 and malate dehyrdrogenase A fragment of MAPKAP-K3 and malate dehyrdrogenase MAPKAP-K3 and a fragment of malate dehyrdrogenase A fragment of MAPKAP-K3 and a fragment of malate dehyrdrogenase

[0042] TABLE 27 Protein Complexes of MAPKAP-K3/GA17 Interaction MAPKAP-K3 and GA17 A fragment of MAPKAP-K3 and GA17 MAPKAP-K3 and a fragment of GA17 A fragment of MAPKAP-K3 and a fragment of GA17

[0043] TABLE 28 Protein Complexes of MAPKAP-K3/Calpain 4 Small Subunit Interaction MAPKAP-K3 and Calpain 4 small subunit A fragment of MAPKAP-K3 and Calpain 4 small subunit MAPKAP-K3 and a fragment of Calpain 4 small subunit A fragment of MAPKAP-K3 and a fragment of Calpain 4 small subunit

[0044] TABLE 29 Protein Complexes of MAPKAP-K3/BAT3 Interaction MAPKAP-K3 and BAT3 A fragment of MAPKAP-K3 and BAT3 MAPKAP-K3 and a fragment of BAT3 A fragment of MAPKAP-K3 and a fragment of BAT3

[0045] TABLE 30 Protein Complexes of MSK-1/AbLim Interaction MSK-1 and abLim A fragment of MSK-1 and abLim MSK-1 and a fragment of abLim A fragment of MSK-1 and a fragment of abLim

[0046] TABLE 31 Protein Complexes of MSK-1/KIAA0144 Interaction MSK-1 and KIAA0144 A fragment of MSK-1 and KIAA0144 MSK-1 and a fragment of KIAA0144 A fragment of MSK-1 and a fragment of KIAA0144

[0047] The involvement of above interactions in particular pathways is as follows.

[0048] Many cellular proteins exert their function by interacting with other proteins in the cell. Examples of this are found in the formation of multiprotein complexes and the association of an enzymes with their substrates. It is widely believed that a great deal of information can be gained by understanding individual protein-protein interactions, and that this is useful in identifying complex networks of interacting proteins that participate in the workings of normal cellular functions. Ultimately, the knowledge gained by characterizing these networks can lead to valuable insight into the causes of human diseases and can eventually lead to the development of therapeutic strategies. The yeast two-hybrid assay is a powerful tool for determining protein-protein interactions and it has been successfully used for studying human disease pathways. In one variation of this technique, a protein of interest (or a portion of that protein) is expressed in a population of yeast cells that collectively contain all protein sequences. Yeast cells that possess protein sequences that interact with the protein of interest are then genetically selected and the identity of those interacting proteins are determined by DNA sequencing. Thus, proteins that can be demonstrated to interact with a protein known to be involved in a human disease are therefore also implicated in that disease. To create a more complex network of interactions in a disease pathway, proteins that were identified in the first round of two-hybrid screening are subsequently used in two-hybrid assays as the protein of interest.

[0049] Cellular events that are initiated by exposure to growth factors, cytokines and stress are propagated from the outside of the cell to the nucleus by means of several protein kinase signal transduction cascades. p38 kinase is a member of the MAP kinase family of protein kinases. It is a key player in signal transduction pathways induced by the proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) and it also plays a critical role in the synthesis and release of the proinflammatory cytokines (Raingeaud et al., 1995; Lee et al., 1996; Miyazawa et al., 1998; Lee et al., 1994). Studies of inhibitors of p38 kinase have shown that blocking p38 kinase activity can cause anti-inflammatory effects, thus suggesting that this may be a mechanism of treating certain inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Further, p38 kinase activity has been implicated in other human diseases such as atherosclerosis, cardiac hypertrophy and hypoxic brain injury (Grammner et al., 1998; Mach et al., 1998; Wang et al., 1998; Nemoto et al., 1998; Kawasaki et al., 1997). Thus, by understanding p38 function, one may gain novel insight into a cellular response mechanism that affects a number of tissues and can potentially lead to harmful affects when disrupted.

[0050] The search for the physiological substrates of p38 kinase has taken a number of approaches including a variety of biochemical and cell biological methods. There are four known human isoforms of p38 kinase termed alpha, beta, gamma and delta, and these are thought to possess different physiological functions, likely because they have distinct substrate and tissue specificities.

[0051] Some of the p38 kinase substrates are known, and the list includes transcription factors and additional protein kinases that act downstream of p38 kinase. Four of the kinases that act downstream of p38 kinase, MAPKAP-K2, MAPKAP-K3, PRAK and MSK1, are currently being analyzed themselves and some of their substrates and regulators have been identified.

[0052] Initial two-hybrid screens have been performed and the search results are now described.

[0053] The yeast two-hybrid system has been used to detect potential substrates and upstream regulators of the p38 kinases and their downstream kinases. In a two-hybrid search using p38 alpha as the protein of interest, an interaction with the guanine nucleotide-exchange protein cytohesin-4 (CYT4) was identified. CYT4 is a member of the PSCD protein family and has a structural organization identical to other PSCD proteins, consisting of an N-terminal coiled-coil motif, a central Sec7 homology domain, and a C-terminal pleckstrin homology (PH) domain. The coiled-coil motif is involved in homodimerization, the Sec7 domain contains guanine-nucleotide exchange protein (GEP) activity, and the PH domain interacts with phospholipids and is responsible for association of PSCD proteins with membranes. Members of this family appear to mediate the regulation of protein sorting and membrane trafficking. CYT4 exhibits GEP activity in vitro with ADP-ribosylation factors ARF1 and ARF5 but is inactive with ARF6 (Ogasawara et al., 2000). CYT4 may act as either a substrate or a regulator of p38 alpha kinase in inflammation or other disease-related signal transduction pathways.

[0054] When the mitogen-activated MAP kinase activator 3pK (MAPKAP-K3) was used in a two-hybrid search, two interactors were identified. The first novel protein, PN2012, bears similarity to the mouse transcription factor Kaiso (GenBank accession AF097416). Kaiso, is a zinc-finger containing protein of the POZ-ZF variety; other related members of this family have been implicated in developmental control and cancer (Daniel et al., 1999). MAPKAP-K3 may phosphorylate this putative transcription factor, thereby altering its activity and affecting the transcription of a set of inflammation-related genes. In support of this hypothesis, Kaiso contains one MAPKAP consensus phosphorylation site.

[0055] The second interactor identified for MAPKAP-K3 is the novel protein PN7771. PN7771 is highly related (greater than 90% amino acid identity) to Ninein. Ninein is a centrosome-associated protein that interacts with human glycogen synthase kinase 3beta (GSK-3beta) (Hong et al., 2000), is localized to the pericentriolar matrix of the centrosome, and reacts with centrosomal autoantibody sera (Mack et al., 1998). PN7771 contains predicted calcium-binding EF hand motifs, a potential nuclear localization signal, a basic region-leucine zipper motif, a spectrin repeat, coiled-coil motifs, and Glu- and Gln-rich regions. The interaction with MAPKAP-K3 suggests PN7771 may be responsive to MAPK signaling pathways, perhaps serving as a substrate for MAPKAP-K3.

[0056] In support of this, we find several MAPKAP consensus phosphorylation sites in PN7771.

[0057] In a two-hybrid search using the p38-regulated protein kinase PRAK, an interaction with the novel protein PN7098 was identified. PN7098 is a 1,231 amino acid polypeptide, although the sequence is incomplete at the 3′ (C-terminal) end. PN7098 contains a PKC C1 (diacylglycerol/phorbol ester-binding) domain, several Ser-rich regions, and two potential nuclear localization signals. PN7098 is related (86% amino acid identity) to the rat Munc13-3 protein (GenBank accession U75361), which is involved in neurotransmitter release (Augustin, et al., 1999) PN7098 may function as either a regulator or a substrate of PRAK protein kinase activity.

[0058] Further two-hybrid screens have also been performed and the search results are now described. In a two-hybrid search using p38 alpha kinase as the protein of interest, four proteins were shown to bind to p38 alpha. The first protein, JNK3 alpha2, is also a serine/threonine protein kinase of the MAP kinase family that is involved in signal transduction (Gupta et al., 1996). Like the p38 kinase pathway constituents, the JNK kinases are activated in response to extracellular stimulation by IL-1. The JNK kinases function by phosphorylating various transcription factors, thereby altering gene expression patterns. The interaction of p38 alpha and JNK3 alpha2 suggests that JNK3 alpha2 is either a substrate or a regulator of p38 alpha, and further identifies a potential link between JNK3 and the inflammatory response. Is further support of such a link, we have subsequently identified yeast two-hybrid interactions between p38 alpha and both JNK1 and JNK2.

[0059] The second protein that interacts with p38 alpha is the large centrosomal protein C-NAP 1. C-NAP1 is a 2,442 amino acid protein that was originally identified by its interaction with the Nek2 cell cycle-regulated protein kinase (Fry et al., 1998). C-NAP1 contains multiple coiled-coiled domains that are likely to be involved in protein-protein interactions. The finding that C-NAP1 interacts with p38 alpha suggests that it is a substrate of both Nek2 and p38 kinases. Thus, C-NAP1 may play a critical role in cellular growth control and in the cellular inflammatory response. Further, by inference, this result links p38 alpha to cellular growth control and Nek2 to inflammation.

[0060] The third p38 alpha-interacting protein, vinculin, resides in the cytoplasmic side of adhesion plaques and may participate in actin microfilament attachment (Rudiger, 1998). Vinculin has been characterized as a tumor suppressor, suggesting that it may play a regulatory function in addition to a structural role in the cell. Vinculin is post-translationally modified by phosphorylation, suggesting it may be a substrate for p38 kinase. Given the requirements for cytoskeletal rearrangement and changes in cell adhesion in the inflammatory response, our results suggest that phosphorylation of vinculin by p38 alpha may be involved in cellular responses to inflammatory stimuli. This interaction is reminiscent of another interaction (see below) between a kinase downstream of p38 (MSK1) and the actin-binding protein ABLIM.

[0061] The fourth p38 alpha-interacting protein was identified with a mutant p38 alpha, in which lysine 53 was changed to a methionine (K53M), rendering the kinase catalytically inactive and presumably stabilizing transient protein-protein interactions. Using this K53M mutant as bait in a two-hybrid assay, the RNA splicing factor PSF was found to be an interactor. PSF is a nuclear protein that contains two RNA recognition motifs and has been found to form a complex with the polypyrimidine tract-binding protein PTB (Patton et al., 1993). Regulation of mRNA splicing is an effective way to modulate protein expression levels, and consequently the interaction of PSF and p38 alpha suggests that phosphorylation of the former by the latter may result in changes in the expression of proteins involved in the inflammatory response. Interestingly, PSF has been shown to bind to the protein phosphatase PP1 delta (Hirano et al., 1996), suggesting a scenario in which PSF activity is controlled by the opposite actions of p38 alpha kinase and PP1 delta phosphatase.

[0062] MAPKAP-K2, a protein kinase that acts downstream of p38 kinase in the same signal transduction pathway, was used in a two-hybrid search to identify potential substrates or regulators. MAPKAP-K2 was demonstrated to interact with five proteins. The first of these is a leucine-rich protein L130. L130 was identified by virtue of its high level of expression in hepatoblastoma cells (Hou et al., 1994). The expression of L 130 in hepatoblastoma cells suggests a role in liver function or in the transformation of normal cells to malignant ones. Interestingly, this protein was also identified as a two-hybrid interactor of another highly related p38-activated protein kinase, PRAK (see below). L130 interacts with the kinase domains of both MAPKAP-K2 and PRAK, suggesting it is a substrate for these kinases. Furthermore, the identification of L130 as an interactor of two kinases involved in the same signaling pathway strongly suggests an important role for L130 in the inflammatory response.

[0063] The second MAPKAP-K2 interactor, cAMP-dependent protein kinase (PKA) regulatory subunit type I alpha, is one component of the PKA serine/threonine protein kinase complex that plays a role in cellular signal transduction. Intracellular levels of cAMP increase in response to various chemical and hormonal stimuli, and PKA is in turn activated by binding to the second messenger cAMP (Francis et al., 1999). The regulatory subunit of PKA is phosphorylated, suggesting PKA may serve as a substrate for MAPKAP-K2. Consistent with this, the region of MAPKAP-K2 that interacts with PKA includes the kinase domain. In addition, we find that that this same subunit of PKA can bind to another p38-activated protein kinase, PRAK (see below). Although the region of PRAK with which PKA interacts does not include the kinase domain, this region of PRAK also interacts with ERK3, another kinase involved in signal transduction. Interestingly, ERK3 also interacts directly with MAPKAP-K2 (see below). Taken together, these results argue that PKA may be involved in the inflammatory response, perhaps as a substrate of these protein kinases.

[0064] Another MAPKAP-K2 interactor involved in signal transduction, ERK3, was found using the MAPKAP-K2 K93M, T222D, T334D triple mutant protein as bait. ERK3 (extracellular signal-regulated protein kinase 3) is a serine/threonine protein kinase (Cheng et al., 1996). It is a nuclear protein presnt in several tissues and is expressed in response to a number of extracellular stimuli.

[0065] Although the biological roles of ERK3 are not yet well understood, it is likely to be part of the MAP kinase cascade initiated in response to pro-inflammatory stimuli. This role is further supported by its interaction with the p38-regulated kinase PRAK; the interactions of ERK3 with both MAPKAP-K2 and PRAK have been confirmed by in vitro assays (see below).

[0066] Another signal transduction protein that binds MAPKAP-K2 is the myeloid leukemia-associated protein SET. SET may be involved in the generation of intracellular signaling events that lead to changes in transcriptional activity after binding of a ligand to HLA class II molecules (Vaesen et al., 1994). In addition, SET is a strong inhibitor of protein phosphatase 2A (Li et al., 1996). Furthermore, SET appears to play a role in cell proliferation, as SET mRNA expression is markedly reduced in cells rendered quiescent by serum starvation, contact inhibition, or differentiation (Carlson et al., 1998). Consistent with a role for SET in growth control and differentiation, fusion of the SET protein with part of the CAN oncogene as the result of a chromosome translocation results in leukemia (von Lindern et al., 1992). SET is a ubiquitously expressed nuclear phosphoprotein that resembles members of the nucleosome assembly protein family. The SET protein is phosphorylated on serine and threonine residues (in addition to tyrosines), suggesting SET may be a substrate of MAPKAP-K2 kinase activity.

[0067] The fourth MAPKAP-K2 interactor is the protein product of the TL21 transcript. In a study designed to examine cDNAs that are differentially expressed between androgen-dependent and androgen-independent prostate carcinoma cell lines, TL21 was isolated as a transcript showing a marked increase in the androgen-dependent cell line (Blok et al., 1995). The TL21 protein product with which MAPKAP-K2 interacts contains no discernible structural motifs, and consequently possible functions of TL21 cannot be deduced. However, the interaction with MAPKAP-K2 suggests it may serve as a substrate or regulator of MAPKAP-K2 kinase activity.

[0068] When a second p38-activated protein kinase, MAPKAP-K3, was used in a two-hybrid search, five proteins were demonstrated to interact with it. The first MAPKAP-K3 interactor is thrombospondin 3, an adhesive glycoprotein that is involved in cell-to-cell and cell-to-matrix interactions (Qabar et al., 1994). It is normally localized extracellularly; however, a number of extracellular proteins exist at low concentrations, or in certain cell types, within the cytoplasm, so we cannot rule out a biological role for the interaction with MAPKAP-K3 in the inflammatory response.

[0069] The second MAPKAP-K3 interactor is malate dehydrogenase, a cytoplasmic enzyme that catalyzes an NAD-dependent reversible reaction of the citric acid cycle (Musrati et al., 1998). The finding that MAPKAP-K3 interacts with this protein suggests the protein kinase cascade that responds to inflammatory stimuli may affect cellular metabolism.

[0070] The third MAPKAP-K3-interacting protein, GA17, has no known function; it is described in the public databases only as a novel gene isolated from human dendritic cells. The only discernible structural feature is a PCI or PINT domain near the C-terminus; this domain is found in proteasome subunits and proteins involved in translation initiation and intracellular signal transduction, but it has no known function. Although functions of this protein are not yet apparent, we infer that it may serve either upstream or downstream of MAPKAP-K3 in the inflammation response pathway.

[0071] The fourth MAPKAP-K3 interactor is the small subunit of the calcium-dependent protease calpain. Calpain is a non-lysosomal calcium-activated thiol-protease composed of large and small subunits; the small subunit with which MAPKAP-K3 interacts possesses regulatory activity. The true biological substrates of calpain are unknown, however a multitude of proteins can act as substrates in vitro (Saido et al., 1994). Interestingly, calpain has been shown to interact with IL-2 receptor gamma chain, and is responsible for cleavage of this protein (Noguchi et al., 1997).

[0072] Furthermore, calpain inhibitors have been shown to interfere with NFKB activation (Kouba et al., 2000), further implicating calpain in intracellular signaling in response to external stimuli. In light of these results, the interaction with MAPKAP-K3 suggests calpain activity may be modulated by MAPKAP-K3 phosphorylation, and that this has an effect on signal transduction in response to inflammatory signals.

[0073] The fifth MAPKAP-K3-interacting protein is BAT3. BAT3 a large proline-rich protein of unknown function that was identified as an HLA-B-associated transcript and was cloned from a human T-cell line (Banedi et al., 1990). BAT3 is a large cytoplasmic protein that is very rich in proline and includes short tracts of polyproline, polyglycine, and charged amino acids. BAT3 transcripts are present in all adult tissues with the highest levels found in testis (Ozaki et al., 1999). BAT3 was demonstrated to bind to a candidate neuroblastoma tumor suppressor, DAN. DAN is a zinc-finger containing protein that may participate in the cell cycle regulation of DNA synthesis. Both DAN and BAT3 are down-regulated in transformed cells. The interaction with MAPKAP-K3 suggests function either upstream or downstream of this kinase in the inflammatory response.

[0074] Another p38-activated protein kinase, MSK-1, was used in a two-hybrid assay and it was found to bind to two proteins. The first, ABLIM, possesses two apparent functional domains: an actin-binding region, and a LIM domain region that is likely involved in protein-protein interactions (Roof et al., 1997). ABLIM may function by coupling the actin-based cytoskeleton to intracellular signaling pathways via its association with MSK-1. This type of function is critical for cell differentiation and morphogenesis, events that occur in response to exposure to external stimuli. This interaction is reminiscent of the interaction between p38 alpha and the cell adhesion/cytoskeleton related protein vinculin, suggesting that phosphorylation of cytoskeletal components may be an important response to inflammatory stimuli.

[0075] MSK-1 has also been demonstrated to interact with KIAA0144, a protein of unknown function. The only discernible structural features of KIAA0144 are Ser-, Pro-, and Thr-rich regions.

[0076] Analysis of homologous ESTs suggests expression in a large variety of tissues. Interaction with MSK-1 suggests function either as a regulator or a substrate of this kinase.

[0077] In a two-hybrid search using the p38-regulated protein kinase PRAK, eleven proteins were identified as PRAK interactors and are therefore implicated in the regulation of inflammatory responses and associated diseases. Two of these proteins, ERK3 and the cAMP-dependent protein kinase (PKA) regulatory subunit, are involved in signal transduction and have been described above as interactors of MAPKAP-K2 in the two-hybrid system. The interaction of ERK3 and PKA with both MAPKAP-K2 and PRAK strengthens the hypothesized role of PKA and ERK3 in the signal transduction cascades that result from inflammatory stimuli.

[0078] PRAK interacts with two proteins thought to be involved in vesicular transport. The first protein, Hook1, was isolated based on sequence similarity to the Drosophila Hook protein. The Drosophila homolog is a cytoplasmic coiled-coil protein that functions in the endocytosis of transmembrane receptors and their ligands from the cell surface to the inside of the cell (Kramer et al., 1996). Human Hook1 may participate in signal transduction by internalizing receptors or ligands involved intercellular communication. The second PRAK interactor involved in intracellular protein transport is golgin-95. Golgin-95 is a coiled-coil protein that localizes to the Golgi apparatus (Fritzler et al., 1993; Barr, 1999). Its precise function is unknown, but interestingly, it has been shown to cross-react with certain human autoimmune sera. The interaction of Hook1 and golgin-95 with PRAK suggests these proteins may be substrates of PRAK protein kinase activity, and that PRAK may cause changes in intracellular transport in response to external signals by modulating the activity of these proteins.

[0079] PRAK also binds proteins that function in transcriptional regulation, immune response and mitosis. PRAK has been demonstrated to interact with the Prox1 transcription factor. Prox1 is a homeobox-containing protein that has been well studied in mice, and it has been shown to be necessary for the development of the mouse lymphatic system (Wigle et al., 1999). PRAK may be capable of phosphorylating Prox1, thereby affecting its transcriptional function. PRAK has been demonstrated to bind to the immunoglobulin gamma heavy chain constant region. Immunoglobulin molecules recognize antigens and are the first step of the immune response. Although immunoglobulin molecules normally reside outside of the cell, it is possible that PRAK or some other related protein kinase could phosphorylate them to affect their function. This interaction may serve as a direct tie between PRAK and the immune response. PRAK has been shown to interact with kendrin, a large centrosomal protein also called pericentrin. Kendrin forms a complex with gamma tubulin and the dynein motor, and likely plays a critical role in the organization of the mitotic spindle (Purohit et al., 1999). PRAK binding to kendrin suggests that kendrin is a substrate of PRAK; thus, PRAK may play an important function the control of chromosome segregation at mitosis. This interaction is reminiscent of the interaction described above between p38 alpha and the centrosomal protein C-NAP 1, and may serve similar functions.

[0080] PRAK has been shown to bind to four proteins for which functions have not yet been determined. The first of these, KIAA0555, was isolated from brain, but analysis of homologous ESTs suggests it is expressed in a variety of tissues. KIAA0555 contains numerous predicted coiled-coil motifs, likely involved in protein-protein interactions, and it displays weak homology (˜20% amino acid identity) to myosin heavy chains from a variety of organisms. We have subsequently identified an interaction between KIAA0555 and protein 14-3-3 epsilon, a member of a large family of proteins involved in signal transduction; the domains with which PRAK and 14-3-3 epsilon interact overlap, suggesting that KIAA0555 may serve as a bridge between PRAK and 14-3-3-dependent signaling pathways. The next PRAK interactor without known function is the leucine-rich protein L130. L130 was described above as an interactor of MAPKAP-K2. Both PRAK and MAPKAP-K2 interact with the same region of L130, arguing that L130 plays in important role in the inflammatory response. The final two PRAK interactors are referred to by their Genbank accession numbers, AL117237 and AL117538. AL117237 was isolated from adult uterus, and analysis of homologous ESTs suggests nearly ubiquitous expression. Analysis of the predicted protein sequence indicates the presence of a coiled-coil region, Arg- and Glu-rich regions, and several nuclear localization signals. AL117538 was isolated from adult testis, and analysis of homologous ESTs suggests expression in a variety of tissues. The predicted protein contains a spectrin repeat and a coiled-coil region. The interaction of these two proteins with PRAK suggests that they may function either as substrates or regulators of the PRAK protein kinase activity and link these two proteins to the inflammatory response and to inflammation-associated diseases.

[0081] The proteins disclosed in the present invention were found to interact with their corresponding proteins in the yeast two-hybrid system. Because of the involvement of the corresponding proteins in the physiological pathways disclosed herein, the proteins disclosed herein also participate in the same physiological pathways. Therefore, the present invention provides a list of uses of these proteins and DNA encoding these proteins for the development of diagnostic and therapeutic tools useful in the physiological pathways. This list includes, but is not limited to, the following examples.

[0082] Two-Hybrid System

[0083] The principles and methods of the yeast two-hybrid system have been described in detail elsewhere (e.g., Bartel and Fields, 1997; Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992). The following is a description of the use of this system to identify proteins that interact with a protein of interest.

[0084] The target protein is expressed in yeast as a fusion to the DNA-binding domain of the yeast Gal4p. DNA encoding the target protein or a fragment of this protein is amplified from cDNA by PCR or prepared from an available clone. The resulting DNA fragment is cloned by ligation or recombination into a DNA-binding domain vector (e.g., pGBT9, pGBT.C, pAS2-1) such that an in-frame fusion between the Gal4p and target protein sequences is created.

[0085] The target gene construct is introduced, by transformation, into a haploid yeast strain. A library of activation domain fusions (i.e., adult brain cDNA cloned into an activation domain vector) is introduced by transformation into a haploid yeast strain of the opposite mating type. The yeast strain that carries the activation domain constructs contains one or more Gal4p-responsive reporter gene(s), whose expression can be monitored. Examples of some yeast reporter strains include Y190, PJ69, and CBY14a. An aliquot of yeast carrying the target gene construct is combined with an aliquot of yeast carrying the activation domain library. The two yeast strains mate to form diploid yeast and are plated on media that selects for expression of one or more Gal4p-responsive reporter genes. Colonies that arise after incubation are selected for further characterization.

[0086] The activation domain plasmid is isolated from each colony obtained in the two-hybrid search. The sequence of the insert in this construct is obtained by the dideoxy nucleotide chain termination method. Sequence information is used to identify the gene/protein encoded by the activation domain insert via analysis of the public nucleotide and protein databases. Interaction of the activation domain fusion with the target protein is confirmed by testing for the specificity of the interaction. The activation domain construct is co-transformed into a yeast reporter strain with either the original target protein construct or a variety of other DNA-binding domain constructs. Expression of the reporter genes in the presence of the target protein but not with other test proteins indicates that the interaction is genuine.

[0087] In addition to the yeast two-hybrid system, other genetic methodologies are available for the discovery or detection of protein-protein interactions. For example, a mammalian two-hybrid system is available commercially (Clontech, Inc.) that operates on the same principle as the yeast two-hybrid system. Instead of transforming a yeast reporter strain, plasmids encoding DNA-binding and activation domain fusions are transfected along with an appropriate reporter gene (e.g., lacZ) into a mammalian tissue culture cell line. Because transcription factors such as the Saccharomyces cerevisiae Gal4p are functional in a variety of different eukaryotic cell types, it would be expected that a two-hybrid assay could be performed in virtually any cell line of eukaryotic origin (e.g., insect cells (SF9), fingal cells, worm cells, etc.). Other genetic systems for the detection of protein-protein interactions include the so-called SOS recruitment system (Aronheim et al., 1997).

[0088] Protein-protein Interactions

[0089] Protein interactions are detected in various systems including the yeast two-hybrid system, affinity chromatography, co-immunoprecipitation, subcellular fractionation and isolation of large molecular complexes. Each of these methods is well characterized and can be readily performed by one skilled in the art. See, e.g., U.S. Pat. Nos. 5,622,852 and 5,773,218, and PCT published applications No. WO 97/27296 and WO 99/65939, each of which are incorporated herein by reference.

[0090] The protein of interest can be produced in eukaryotic or prokaryotic systems. A cDNA encoding the desired protein is introduced in an appropriate expression vector and transfected in a host cell (which could be bacteria, yeast cells, insect cells, or mammalian cells). Purification of the expressed protein is achieved by conventional biochemical and immunochemical methods well known to those skilled in the art. The purified protein is then used for affinity chromatography studies: it is immobilized on a matrix and loaded on a column. Extracts from cultured cells or homogenized tissue samples are then loaded on the column in appropriate buffer, and non-binding proteins are eluted. After extensive washing, binding proteins or protein complexes are eluted using various methods such as a gradient of pH or a gradient of salt concentration. Eluted proteins can then be separated by two-dimensional gel electrophoresis, eluted from the gel, and identified by micro-sequencing. The purified proteins can also be used for affinity chromatography to purify interacting proteins disclosed herein. All of these methods are well known to those skilled in the art.

[0091] Similarly, both proteins of the complex of interest (or interacting domains thereof) can be produced in eukaryotic or prokaryotic systems. The proteins (or interacting domains) can be under control of separate promoters or can be produced as a fusion protein. The fusion protein may include a peptide linker between the proteins (or interacting domains) which, in one embodiment, serves to promote the interaction of the proteins (or interacting domains). All of these methods are also well known to those skilled in the art.

[0092] Purified proteins of interest, individually or a complex, can also be used to generate antibodies in rabbit, mouse, rat, chicken, goat, sheep, pig, guinea pig, bovine, and horse. The methods used for antibody generation and characterization are well known to those skilled in the art. Monoclonal antibodies are also generated by conventional techniques. Single chain antibodies are further produced by conventional techniques.

[0093] DNA molecules encoding proteins of interest can be inserted in the appropriate expression vector and used for transfection of eukaryotic cells such as bacteria, yeast, insect cells, or mammalian cells, following methods well known to those skilled in the art. Transfected cells expressing both proteins of interest are then lysed in appropriate conditions, one of the two proteins is immunoprecipitated using a specific antibody, and analyzed by polyacrylamide gel electrophoresis. The presence of the binding protein (co-immunoprecipitated) is detected by immunoblotting using an antibody directed against the other protein. Co-immunoprecipitation is a method well known to those skilled in the art.

[0094] Transfected eukaryotic cells or biological tissue samples can be homogenized and fractionated in appropriate conditions that will separate the different cellular components. Typically, cell lysates are run on sucrose gradients, or other materials that will separate cellular components based on size and density. Subcellular fractions are analyzed for the presence of proteins of interest with appropriate antibodies, using immunoblotting or immunoprecipitation methods. These methods are all well known to those skilled in the art.

[0095] Disruption of Protein-protein Interactions

[0096] It is conceivable that agents that disrupt protein-protein interactions can be beneficial in many physiological disorders, including, but not-limited to NIDDM, AD and others disclosed herein. Each of the methods described above for the detection of a positive protein-protein interaction can also be used to identify drugs that will disrupt said interaction. As an example, cells transfected with DNAs coding for proteins of interest can be treated with various drugs, and co-immunoprecipitations can be performed. Alternatively, a derivative of the yeast two-hybrid system, called the reverse yeast two-hybrid system (Leanna and Hannink, 1996), can be used, provided that the two proteins interact in the straight yeast two-hybrid system.

[0097] Modulation of Protein-protein Interactions

[0098] Since the interaction described herein is involved in a physiological pathway, the identification of agents which are capable of modulating the interaction will provide agents which can be used to track the physiological disorder or to use as lead compounds for development of therapeutic agents. An agent may modulate expression of the genes of interacting proteins, thus affecting interaction of the proteins. Alternatively, the agent may modulate the interaction of the proteins. The agent may modulate the interaction of wild-type with wild-type proteins, wild-type with mutant proteins, or mutant with mutant proteins. Agents can be tested using transfected host cells, cell lines, cell models or animals, such as described herein, by techniques well known to those of ordinary skill in the art, such as disclosed in U.S. Pat. Nos. 5,622,852 and 5,773,218, and PCT published applications No. WO 97/27296 and WO 99/65939, each of which are incorporated herein by reference. The modulating effect of the agent can be screened in vivo or in vitro. Exemplary of a method to screen agents is to measure the effect that the agent has on the formation of the protein complex.

[0099] Mutation Screening

[0100] The proteins disclosed in the present invention interact with one or more proteins known to be involved in a physiological pathway, such as in NIDDM, AD or pathways described herein. Mutations in interacting proteins could also be involved in the development of the physiological disorder, such as NIDDM, AD or disorders described herein, for example, through a modification of protein-protein interaction, or a modification of enzymatic activity, modification of receptor activity, or through an unknown mechanism. Therefore, mutations can be found by sequencing the genes for the proteins of interest in patients having the physiological disorder, such as insulin, and non-affected controls. A mutation in these genes, especially in that portion of the gene involved in protein interactions in the physiological pathway, can be used as a diagnostic tool and the mechanistic understanding the mutation provides can help develop a therapeutic tool.

[0101] Screening for At-risk Individuals

[0102] Individuals can be screened to identify those at risk by screening for mutations in the protein disclosed herein and identified as described above. Alternatively, individuals can be screened by analyzing the ability of the proteins of said individual disclosed herein to form natural complexes. Further, individuals can be screened by analyzing the levels of the complexes or individual proteins of the complexes or the mRNA encoding the protein members of the complexes. Techniques to detect the formation of complexes, including those described above, are known to those skilled in the art. Techniques and methods to detect mutations are well known to those skilled in the art.

[0103] Techniques to detect the level of the complexes, proteins or mRNA are well known to those skilled in the art.

[0104] Cellular Models of Physiological Disorders

[0105] A number of cellular models of many physiological disorders or diseases have been generated. The presence and the use of these models are familiar to those skilled in the art. As an example, primary cell cultures or established cell lines can be transfected with expression vectors encoding the proteins of interest, either wild-type proteins or mutant proteins. The effect of the proteins disclosed herein on parameters relevant to their particular physiological disorder or disease can be readily measured. Furthermore, these cellular systems can be used to screen drugs that will influence those parameters, and thus be potential therapeutic tools for the particular physiological disorder or disease. Alternatively, instead of transfecting the DNA encoding the protein of interest, the purified protein of interest can be added to the culture medium of the cells under examination, and the relevant parameters measured.

[0106] Animal Models

[0107] The DNA encoding the protein of interest can be used to create animals that overexpress said protein, with wild-type or mutant sequences (such animals are referred to as “transgenic”), or animals which do not express the native gene but express the gene of a second animal (referred to as “transplacement”), or animals that do not express said protein (referred to as “knock-out”). The knock-out animal may be an animal in which the gene is knocked out at a determined time. The generation of transgenic, transplacement and knock-out animals (normal and conditioned) uses methods well known to those skilled in the art.

[0108] In these animals, parameters relevant to the particular physiological disorder can be measured. These parametes may include receptor function, protein secretion in vivo or in vitro, survival rate of cultured cells, concentration of particular protein in tissue homogenates, signal transduction, behavioral analysis, protein synthesis, cell cycle regulation, transport of compounds across cell or nuclear membranes, enzyme activity, oxidative stress, production of pathological products, and the like. The measurements of biochemical and pathological parameters, and of behavioral parameters, where appropriate, are performed using methods well known to those skilled in the art. These transgenic, transplacement and knock-out animals can also be used to screen drugs that may influence the biochemical, pathological, and behavioral parameters relevant to the Be particular physiological disorder being studied. Cell lines can also be derived from these animals for use as cellular models of the physiological disorder, or in drug screening.

[0109] Rational Drug Design

[0110] The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. Several approaches for use in rational drug design include analysis of three-dimensional structure, alanine scans, molecular modeling and use of anti-id antibodies. These techniques are well known to those skilled in the art.

[0111] Following identification of a substance which modulates or affects polypeptide activity, the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.

[0112] A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.

[0113] The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This approach might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.

[0114] Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a target property.

[0115] Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.

[0116] A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted thereon can be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

[0117] Diagnostic Assays

[0118] The identification of the interactions disclosed herein enables the development of diagnostic assays and kits, which can be used to determine a predisposition to or the existence of a physiological disorder. In one aspect, one of the proteins of the interaction is used to detect the presence of a “normal” second protein (i.e., normal with respect to its ability to interact with the first protein) in a cell extract or a biological fluid, and further, if desired, to detect the quantitative level of the second protein in the extract or biological fluid. The absence of the “normal” second protein would be indicative of a predisposition or existence of the physiological disorder. In a second aspect, an antibody against the protein complex is used to detect the presence and/or quantitative level of the protein complex. The absence of the protein complex would be indicative of a predisposition or existence of the physiological disorder.

[0119] Nucleic Acids and Proteins

[0120] A nucleic acid or fragment thereof has substantial identity with another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases. A protein or fragment thereof has substantial identity with another if, optimally aligned, there is an amino acid sequence identity of at least about 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity.

[0121] Identity means the degree of sequence relatedness between two polypeptide or two polynucleotides sequences as determined by the identity of the match between two strings of such sequences, such as the full and complete sequence. Identity can be readily calculated. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Methods commonly employed to determine identity between two sequences include, but are not limited to those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J Applied Math. 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Such methods are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG (Genetics Computer Group, Madison Wis.) program package (Devereux, J., et al., Nucleic Acids Research 12(1). 387 (1984)), BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)). The well-known Smith Waterman algorithm may also be used to determine identity.

[0122] Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement. Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Asubel, 1992; Wetmur and Davidson, 1968.

[0123] The terms “isolated”, “substantially pure”, and “substantially homogeneous” are used interchangeably to describe a protein or polypeptide which has been separated from components which accompany it in its natural state. A monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification.

[0124] Large amounts of the nucleic acids of the present invention may be produced by (a) replication in a suitable host or transgenic animals or (b) chemical synthesis using techniques well known in the art. Constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell. Such vectors may be prepared by means of standard recombinant techniques well known in the.

EXAMPLES

[0125] The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.

Example 1 Yeast Two-Hybrid System

[0126] The principles and methods of the yeast two-hybrid systems have been described in detail (Bartel and Fields, 1997). The following is thus a description of the particular procedure that we used, which was applied to all proteins.

[0127] The cDNA encoding the bait protein was generated by PCR from brain cDNA. Gene-specific primers were synthesized with appropriate tails added at their 5′ ends to allow recombination into the vector PGBTQ. The tail for the forward primer was 5′-GCAGGAAACAGCTATGACCATACAGTCAGCGGCCGCCAcC-3¹ (SEQ ID NO:1) and the tail for the reverse primer was 5′-ACGGCCAGTCGCGTGGAGTGTTATGTCATGCGGCCGCTA-3′ (SEQ ID NO:2). The tailed PCR product was then introduced by recombination into the yeast expression vector pGBTQ, which is a close derivative of pGBTC (Bartel et al., 1996) in which the polylinker site has been modified to include M13 sequencing sites. The new construct was selected directly in the yeast J693 for its ability to drive tryptophane synthesis (genotype of this strain: Mat α, ade2, his3, leu2, trpl, URA3::GAL1-lacZ LYS2::GAL1-HIS3 gal4del gal80del cyhR2). In these yeast cells, the bait is produced as a C-terminal fusion protein with the DNA binding domain of the transcription factor Gal4 (amino acids 1 to 147). A total human brain (37 year-old male Caucasian) cDNA library cloned into the yeast expression vector pACT2 was purchased from Clontech (human brain MATCHMAKER cDNA, cat. # HL4004AH), transformed into the yeast strain J692 (genotype of this strain: Mat a, ade2, his3, leu2, trpl, URA3::GAL1-lacZ LYS2::GAL1-HIS3 gal4del gal80del cyhR2), and selected for the ability to drive leucine synthesis. In these yeast cells, each cDNA is expressed as a fusion protein with the transcription activation domain of the transcription factor Gal4 (amino acids 768 to 881) and a 9 amino acid hemagglutinin epitope tag. J693 cells (Mat a type) expressing the bait were then mated with J692 cells (Mat a type) expressing proteins from the brain library. The resulting diploid yeast cells expressing proteins interacting with the bait protein were selected for the ability to synthesize tryptophan, leucine, histidine, and β-galactosidase. DNA was prepared from each clone, transformed by electroporation into E. coli strain KC8 (Clontech KC8 electrocompetent cells, cat. # C2023-1), and the cells were selected on ampicillin-containing plates in the absence of either tryptophane (selection for the bait plasmid) or leucine (selection for the brain library plasmid). DNA for both plasmids was prepared and sequenced by di-deoxynucleotide chain termination method. The identity of the bait cDNA insert was confirmed and the cDNA insert from the brain library plasmid was identified using BLAST program against public nucleotides and protein databases. Plasmids from the brain library (preys) were then individually transformed into yeast cells together with a plasmid driving the synthesis of lamin fused to the Gal4 DNA binding domain. Clones that gave a positive signal after galactosidase assay were considered false-positives and discarded. Plasmids for the remaining clones were transformed into yeast cells together with plasmid for the original bait. Clones that gave a positive signal after -galactosidase assay were considered true positives.

Example 2 Identification of p38 Alpha/CYT4 Interaction

[0128] A yeast two-hybrid system as described in Example 1 using amino acids 194-319 of p38 alpha (Swiss Protein (SP) accession no. Q13083) as bait was performed. One clone that was identified by this procedure included amino acids 4-218 of CYT4 (GenBank (GB) accession no. AF075458).

Example 3 Identification of MAPKAP-K3/PN2012 Interaction

[0129] A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 92-1003 of MAPKAP-K3 (GB accession no. U9578) as bait was performed. One clone that was identified by this procedure included novel protein PN2012. The DNA sequence and the predicted protein sequence for PN2012 are set forth in Tables 32 and 33, respectively. The start codon and stop codon are bolded in Table 32. Several variants were also found, including:

[0130] T is substituted for C at nucleotide position 1190,

[0131] C is subsituted for T as nucleotide position 2839,

[0132] A is substituted for G at nucleotide position 3338,

[0133] G is substitued for A at nucleotide position 4753, and

[0134] nucleotides at positions 723-725 are deleted (also underlined in Table 32). TABLE 32 Nucleotide Sequence ot PN2012 gccgcgtcgacgtcgacccagactggagcgacgtttaaagaaggggcagaatcgctggggagtgcggcttcttcttgttgggggactcccagc cttccgcgcgtccggaggaggagaagcggcggcgccgggaagcaggcatggagagtagaaaactgatttctgctacagacattcagtactct ggcagtctgctgaactccttgaatgagcaacgtggccatggactcttctgtgatgttaccgttattgtggaagaccgaaaattccgggctcacaaga tggtgtcccattgtcacaggttaaaagcatctcaggtacagcgcaggatggtaatactgagcctttacctcctgattctggtgacaagaaccttgtaa tacagaaatcaaaagatgaagcccaagataatggggctactataatgcctattataacagagtctttttcattatctgccgaagattatgaaatgaaaa agatcattgttaccgattctgatgatgatgatgatgatgatgtcattttttgctccgagattctgcccacaaaggagactttgccgagtaataacacagt ggcacaggtccaatctaacccaggccctgttgctatttcagatgttgcacctagtgctagcaataactcgccccctttaacaaatatcacacctactc agaaacttcctactcctgtgaatcaggcaactttgagccaaacacaaggaagtgaaaaattgttggtatcttcagctccaacacatctgactcccaat agacaccaaacagtgctattttaacaggaaacaaggccaatgaagaggaggaggaggaaataatagatgatgatgatgacactattagctccag tcctgactcggccgtcagtaatacatctttggtcccacaggctgatacctcccaaaataccagttttgatggatcattaatacagaagatgcagattcc ggtcagaagatcattactttagatacagctactgaaattgaaggcttatcgactggttgcaaggtttatgcaaatatcggtgaagatacttatgatata gtgatccctgtcaaagatgaccctgatgaaggggaggccagacttgagaatgaaataccaaaaacgtctggcagcgagatggcaaacaaacgt tgcggagacattttaacattcattcttgggagaagaagtatccgtgccgttactgtgagaaggtatttcctcttgcagaatatcgcacaaagcatgaa attcatcacacaggggagcgaaggtatcagtgtttggcctgtggcaaatctttcatcaactatcagtttatgtcttcacatataaagtcagttcatagtc gcaatgaaggatgatggtattgggtataaggttgacactggaaaagaacctccagtagggaccactacatctactcagaacaagccaatgacctg ggaagatatttttattcagcaggaaaatgattcaatttttaaacaaaatgtaacagatggcagtactgagtttgaatttataataccagagtcttactaa actcctttgaaatactagaaagttttgttttggatgatggggcaggggtttcagaagatctgtaaaacaaattaaggtgcgaacaagttaatttgatct gccacattatctgaaggaagtgtagtgggatttttgttgataatttttagaagcaaattttcctgaaagttttgagtagaggtgagaccccctccccaag tatctgtttatatagttagttttcagctcatttaaaagaggcaaaaattaaaagcttggagagatagtttcctgaatagaatttgaagcagtctgaatgttc tgctagtccaagcgaacagcaacctcctgctaccctccctctatgaaaatagccatgcagacaagtctctcatctgaagaacaaattagatttagcta aatgaaaacaatctctgtaatggcagataggaggagatgaaaagttctgttgcatggatttttaattctctggctaccacatagtagagaatggaatg cacatatttaaacacttagaaaataaagttaacacttactgaagtgctagtactaaactgtgctagtactaaaagaaaacaggttggaacatacatata cgatgtgaatgatacggatgccggcagagcttccagatctttcagactcaactgctaggtcaattagtttgtcataataaaacttggcagattctacaa tgaatatgacatttaaactctgtgcctactaaaggtatcttctggagtttttgggaggagagaaactggaaaattaaattgtatttttgccagaagactct cacacacaaccactatgaagaaataatgccgcatttttcccccattgtaccaaaaagataaaaaaatggtaaacactgatcaaggtattttgtattgtc atttaattctgaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO:3)

[0135] TABLE 33 Predicted Amino Acid Sequence of PN2012 MESRKLISATDIQYSGSLLNSLNEQRGHGLFCDVTVIVEDRKFRAHKNILSASSTYFHQLFSV AGQVVELSFIRAEIFAEILNYIYSSKIVRVRSDLLDELIKSGQLLGVKFIAELGVPLSQVKSISG TAQDGNTEPLPPDSGDKNLVIQKSKDEAQDNGATIMPIITESFSLSAEDYEMKKIVTDSDDD DDDDVIFCSEILPTKETLPSNNTVAQVQSNPGPVAJSDVAPSASNNSPPLTMTPTQKIPTPVN QATLSQTQGSEKLLVSSAPTHLTPNJILLNQTPLSTPPNVSSSLPNIIMPSSTNLLVQNQQTPNS AILTGKKANEEEEEEIIDDDDDTISSSPDSAVSNTSLMPQADTSQNTSFDGSLIQKMQJPTLLQ EPLSNSLKTSDIITRNTNDPGVGSKHLMEGQKIITLDTATEIEGLSTGCKVYAMGEDTYDIVTP VKDDPDEGEARLENEIPKTSGSEMANKRMKVKHDDHYELIVDGRVYYICIVCKRSYVCLTS LRRHFNIHSWEKKYPCRYCEKVFPLAEYRTKHEIHHTGERRYQCLACGKSFINYQFMSSHIK SVHSQDPSGDSKLYRLHPCRSLQIRQYAYLSDRSSTIPAMIKDDGIGYKVDTGKFPPVGTTTS TQNKIPMTWEDTFIQQIENDSIFKQNVTDGSTEFEFIIPESY (SEQ ID NO:4)

Example 4 Identification of MAPKAP-K3/PN7771 Interaction

[0136] A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides433-1003 of MAPKAP-K3 (GB accession no. U9578) as bait was performed. One clone that was identified by this procedure included novel protein fragment PN7771. The DNA sequence and the predicted protein sequence for PN7771 are set forth in Tables 34 and 35, respectively. TABLE 34 Nucleotide Sequence of PN7771 cttattttgaaaacatttacatagtgattagttaacccaacagaccaatcctgggaagacagccagagcctgcagcaccttagtaacagaaaaactg ataattaggagaagagacctgtccaagaccaggaacctggaccaaaattgtgccatgttgctttactttaatgagtggccccagtaaaaactgagct ctgctttttttatagtcaaaggactggttctgagagccttgttgcagatggctgaggtcaccgtcccaagggtgtatgtcgtgtttggcatccattgcat catggcgaaggcatcttcagatgtgcaggtttcaggctttcatcggaaaatccagcacgttaaaaatgaactttgccacatgttgagcttggaggag gtggccccagtgctgcagcagacattacttcaggacaacctcttgggcagggtacattttgaccaatttaaagaagcattaatactcatcttgtccag aactctgtcaaatgaagaacactttcaagaaccagactgctcactagaagctcagcccaaatatgttagaggtgggaagcgttacggacgaaggt ccttgcccgagttccaagagtccgtggaggagtttcctgaagtgacggtgattgagccactggatgaagaagcgcggccttcacacatcccagc cggtgactgcagtgagcactggaagacgcaacgcagtgaggagtatgaagcggaaggccagttaaggttttggaacccagatgacttgaatgc ttcacagagtggatcttcccctccocaagactggatagaagagaaactgcaagaagtttgtgaagatttggggatcacccgtgatggtcacctgaa ccggaagaagctggtctccatctgtgagcagtatggtttacagaatgtggatggagagatgctcgaggaagtattccataatcttgatcctgacggt catgcagtctttcgatgagagtggacgacgtaccacaacctcatcagcaatgacaagtaccattggctttcgggtcttctcctgcctggatgatggg atgggccatgcatctgtggagagaatactggacacctggcaggaagagggcattgagaacagccaggagatcctgaaggcottggatttcagc ctcgatggaaacatcaatttgacagaattaacactggcccttgaaaatgaacttttggttaccaagaacagcattcaccaggcggctctggccagct ttaaggctgaaatccggcatttgttggaacgagttgatcaggtggtcagagaaaaagagaagctacggtcagatctggacaaggccgagaagct caagtctttaatggcctcggaggtggatgatcaccatgcggccatagagcggcggaatgagtacaacctcaggaaactggatggagagtacaa ggagcgaatagcagccttaaaaaatgaactccgaaaagagagagagcagatcctgcagcaggcaggcaagcagcgtttagaacttgaacagg aaattgaaaaggcaaaaacagaagagaactatatccgggaccgccttgccctctctttaaaggaaaacagtcgtctggaaaatgagcttctagaa aatgcagagaagttggcagaatatgagaatctgacaaacaaacttcagagaaatttggaaaatgtgttagcagaaaagtttggtgacctcgatcct agcagtgctgagttcttcctgcaagaagagagactgacacagatgagaaatgaatatgagcggcagtgcagggtactacaagaccaagtagatg aactccagtctgagctggaagaatatcgtgcacaaggcagagtgctcaggcttccgttgaagaactcaccgtcagaagaagttgaggctaacag cggtggcattgagcccgaacacgggctcggttctgaagaatgcaatccattgaatatgagcattgaggcagagctggtcattgaacagatgaaag aacaacatcacagggacatatgttgcctcagactggagctcgaagataaagtgcgccattatgaaaagcagctggacgaaaccgtggtcagctg caagaaggcacaggagaacatgaagcaaaggcatgagaacgaaacgcgcaccttagaaaaacaaataagtgaccttaaaaatgaaattgctga acttcaggggcaagcagcagtgctcaaggaggcacatcatgaggccacttgcaggcatgaggaggagaaaaaacaactgcaagtgaagcttg aggaggaaaagactcacctgcaggagaagctgaggctgcaacatgagatggagctcaaggctagactgacacaggctcaagcaagctttgag cgggagagggaaggccttcagagtagcgcctggacagaagagaaggtgagaggcttgactcaggaactagagcagtttcaccaggagcagc tgacaagcctggtggagaaacacactcttgagaaagaggagttaagaaaagagctcttggaaaagcaccaaagggagcttcaggagggaagg gaaaaaatggaaacagagtgtaatagaagaacctctcaaatagaagcccagtttcagtctgattgtcagaaagtcactgagaggtgtgaaagcgc tctgcaaagcctggaggggcgctaccgccaagagctgaaggacctccaggaacagcagcgtgaggagaaatcccagtgggaatttgagaag gacgagctcacccaggagtgtgcggaagcccaggagctgctgaaagagactcttaagagagagaaaacaacttctctggtcctgacccagga gagagagatgctggagaaaacatacaaagaacatttgaacagcatggtcgtcgagagacagcagctactccaagacctggaagacctaagaaa tgtatctgaaacccagcaaagcctgctgtctgaccagatacttgagctgaagagcagtcacaaaagggaactgagggagcgtgaggaggtcct gtgccaggcaggggcttcggagcagctggccagccagcggctggaaagactagaaatggaacatgaccaggaaaggcaggaaatgatgtcc aagcttctagccatggagaacattcacaaagcgacctgtgagacagcagatcgagaaagagccgagatgagcacagaaatctccagacttcag agtaaaataaaggaaatgcagcaggcaacatctcctctctcaatgcttcagagtggttgccaggtgataggagaggaggaggtggaaggagatg gagccctgtccctgcttcagcaaggggagcagctgttggaagaaaatggggacgtcctcttaagcctgcagagagctcatgaacaggcagtga aggaaaatgtgaaaatggctactgaaatttctagattgcaacagaggctacaaaagttagagccagggttagtaatgtcttcttgtttggatgagcca gctactgagttttttggaaatactgcggaacaaacagagcagtttttacagcaaaaccgaacgaagcaagtagaaggtgtgaccaggcggcatgt cctaagtgacctggaagatgatgaggtccgggacctgggaagtacagggacgagctctgttcagagacaggaagtcaaaatagaggagtctga agcttcagtagagggtttttctgagcttgaaaacagtgaagagaccaggactgaatcctgggagctgaagaatcagattagtcagcttcaggaaca gctaatgatgttatgtgcggactgtgatcgagcttctgaaaagaaacaggacctactttttgatgtttctgtgctaaaaaagaaactgaagatgcttga gagaatccctgaggcttctcccaaatataagctgttgtatgaagatgtgagccgagaaaatgactgccttcaggaagagctgagaatgatggaga cacgctacgatgaggcactagaaaataacaaagaactcactgcagaggttttcaggttgcaggatgagctgaagaaaatggaggaagtcactga aacattcctcagcctggaaaagagttacgatgaggtcaaaatagaaaatgaggggctgaatgttctggttttgagacttcaaggcaagattgagaa gcttcaggaaagcgtggtccagcggtgtgactgctgcttatgggaagccagtttagagaacctggaaatcgaacctgatggaaatatactccagc tcaatcagacactggaagagtgtgtgcccagggttaggagtgtacatcatgtcatagaggaatgtaagcaagaaaaccagtaccttgaggggaa cacacagctcttggaaaaagtaaaagcacatgaaattgcctggttacatggaacaattcagacacatcaagaaaggccaagagtacagaatcaa gttatactggaggaaaacactactctcctaggctttcaagacaaacattttcagcatcaggccaccatagcagagttagaactggagaaaacaaag ttacaggagctgactaggaagttgaaggagagagtcactattttagttaagcaaaaagatgtactttctcacggagaaaaggaggaagagctgaa ggcaatgatgcatgacttgcagatcacgtgcagtgagatgcagcaaaaagttgaacttctgagatatgaatctgaaaagcttcaacaggaaaattc tattttgagaaatgaaattactactttaaatgaagaagatagcatttctaacctgaaattagggacattaaatggatctcaggaagaaatgtggcaaaa aacggaaactgtaaaacaagaaaatgctgcagttcagaagatggttgaaaatttaaagaaacagatttcagaattaaaaatcaaaaaccaacaatt ggatttggaaaatacagaacttagccaaaagaactctcaaaaccaggaaaaactgcaagaacttaatcaacgtctaacagaaatgctatgccaga aggaaaaagagccaggaaacagtgcattggaggaacgggaacaagagaagtttaatctgaaagaagaactggaacgttgtaaagtgcagtcct ccactttagtgtcttctctggaggcggagctctctgaagttaaaatacagacccatattgtgcaacaggaaaaccaccttctcaaagatgaactgga gaaaatgaaacagctgcacagatgtcccgatctctctgacttccagcaaaaaatctctagtgttctaagctacaacgaaaaactgctgaaagaaaa ggaagctctgagtgaggaattaaatagctgtgtcgataagttggcaaaatcaagtcttttagagcatagaattgcgacgatgaagcaggaacagaa atcctgggaacatcagagtgcgagcttaaagtcacagctggtggcttctcaggaaaaggttcagaatttagaagacaccgtgcagaatgtaaacc tgcaaatgtcccggatgatatctgacctacgagtgactcagcaggaaaaggaggctttaaaacaagaagtgatgtctttacataagcaacttcaga atgctggtggcaagagctgggccccagagatagctactcatccatcagggctccataaccagcagaaaaggctgtcctgggacaagttggatca tctgatgaatgaggaacagcagctgctttggcaagagaatgagaggctccagaccatggtacagaacaccaaagccgaactcacgcactcccg ggagaaggtccgtcaattggaatccaatcttcttcccaagcaccaaaaacatctaaacccatcaggtaccatgaatcccacagagcaagaaaaatt gagcttaaagagagagtgtgatcagtttcagaaagaacaatctcctgctaacaggaaggtcagtcagatgaattcccttgaacaagaattagaaac aattcatttggaaaatgaaggcctgaaaaagaaacaagtaaaactggatgagcagctcatggagatgcagcacctgaggtccactgcgacgcct agcccgtcccctcatgcttgggatttgcagctgctccagcagcaagcctgtccgatggtgcccagggagcagtttctgcagcttcaacgccagct gctgcaggcagaaaggataaaccagcacctgcaggaggaacttgaaaacaggacctccgaaaccaacacaccacagggaaaccaggaaca actggtaactgtcatggaggaacgaatgatagaagttgaacagaaactgaaactagtgaaaaggcttcttcaagagaaagtgaatcagctcaaag aacaactctgcaagaacactaaggcagacgcaatggtgaaggacttgtatgttgaaaatgcccagttgttgaaagctctggaagtgactgaacag cgacagaaaacagcagagaagaaaaattacctcctggaggagaagattgccagcctcagtaatatagttaggaatctgacaccagcgccattga taattggttaggaggcagggcttattaagtggttattaaccgctgacatcagacaaacccaaatctgtagaattctaacctcctaacacctgtgacag aaaagtttcatttggggagctggtcttctaagaaacggataaagccacataattaaagcagttgaactagagggaaagcactgaacaaaccacttt catgagacaataatccgaaaagttcgctttgatatattcctggagggccaagcccatctatttacaaaaggtgaacagcaaaatcaagcactgcttt atgggcaggaacacaagagaaagcaaactgcccaagaagtcatcatgtcagaaactcaatctcaacaaaataatttccatcagggaacttcagg gtttcttgggggcttatgagtctcaccggtcaacccaggaggcctcactacaagagccttgacaaggcactgttttttgtgggactgggagttcaca gagaaataccatacagataccttatatgtatgcatttgtgcaacaatttttgagaaggtgagtggcaatttataatttagttggcaatttataatagaact aaagggaatcaagtttgcctttgagataatacgttacactaagaaaaggaaaatgtggatagtaaaacccacctctctcatcctattgtactctcttct gctttttagaagcctgcacttaagcttagatttgtgaagggagagtagaaggggagaagtagaaccacagtgttttatttatttttctaaaactcttact attggaatgatataattatacaagtaatgccaaaaaccaagtcaaagcctaattaaccaaagcactcatttaaaaatcatcatgtttggacctatctgg attttgcaaacatgaaaaaaaaaaaaaaaaa (SEQ ID NO:5)

[0137] TABLE 35 Predicted Amino Acid Sequence of PN7771 MAEVTVPRVYVVFGIHCIMAKASSDVQVSGFHRKIQHVKNELCHMLSLEEVAPVLQQTLL QDNLLGRVHFDQFKEALILILSRTLSNEEHFQEPDCSLEAQPKYVRGGKRYGRRSLPEFQES VEEFPEVTVIEPLDEEARPSHIPAGDCSEHWKTQRSEEYEAEGQLRFWNPDDLNASQSGSSP PQDWIEEKLQEVCEDLGITRDGHLNRKKLVSICEQYGLQNVDGEMLEEVFJINLDPDGTMS VEDFFYGLFKINGKSLTPSASTPYRQLKRIILSMQSFDESGRRTTTSSAMTSTJGFRVFSCLDD GMGHASVERLLDTWQEEGIENSQEILKALDFSLDGNINLTELTLALENELLVTKNSIIIQAAL ASFKAEIRHLLERVDQVVREKEKLRSDLDKAEKLKSLMASEVDDHHAAIERRNEYNLRKL DGEYKERIAALKNELRKEREQJLQQAGKQRLELEQEIEKAKTEENYIRDRLALSLKIENSRLE NELLENAEKLAEYENLTNKLQRNLENYLAEKFGDLDPSSAEFFLQEERLTQMRNEYERQCR VLQDQVDELQSELEEYRAQGRVLRLPLKNSPSEEVEANSGGIEPEHGLGSEECNPLNMSIEA ELVIEQMKEQHHRDICCLRLELEDKVRHYEKQLDETVVSCKKAQENMKQRHENETRTLEK QISDLKNEIAELQGQAAVLKEAHHEATCRHEEEKKQLQVKJEEEKTHLQEKLRLQHEMEL KARLTQAQASFEREREGLQSSAWTEEKVRGLTQELEQFHQEQLTSLVEKHTLKEELRKEL LEKHQRELQEGREKMETECNRRTSQIEAQFQSDCQKVTERCESALQSLEGRYRQELKDLQE LQSGCQVIGEEEVEGDGALSLLQQGEQLLEENGDVLLSLQRAHEQAVKENVKMATEISRLQ QRLQKLEPGLVMSSCLDEPATEFFGNTAEQTEQFLQQNRTKQVEGVTRRHVLSDLEDDEVR DLGSTGTSSVQRQEVKIEESEASVEGFSELENSEETRTESWELKNQISQLQEQLMMLCADCD RASEKKQDLLFDVSVLKKKLKMLERIPEASPKYKLLYEDVSRENDCLQEELRMMETRYDE ALENNKELTAEVFRLQDELKKMEEVTETFLSLEKSYDEVKIENEGLNVLVLRLQGKIEKLQE DLGSTGTSSVQRQEVKIEESEASVEGFSELENSEETRTESWELKNQISQLQEQLMMLCADCD RASEKKQDLLFDVSVLKKKLKMLERIPEASPKYKLLYEDVSRENDCLQEELRMMETRYDE ALENNKELTAEVFRLQDELKKMEEVTETFLSLEKSYDEVKIENEGLNVLVLRLQGKIEKLQE SVVQRCDCCLWEASLENLEIEPDGNILQLNQTLEECVPRVRSVTIIWIEECKQENQYLEGNT QLLEKVKAHEIAWLHGTJQTHQERPRVQNQVILEENTTLLGFQDJQIFQHQATIAELELEKTK LQELTRKLKERVTILVKQKDVLSHGEKEEELKAMMHDLQITCSEMQQKVELLRYESEKLQ QENSILRNEITTNEEDSISNLKLGTLNGSQEEMWQKTETVKQENAAVQKMVENLKKQISE LKIKNQQLDLENTELSQKNSQNQEKLQELNQRTEMLCQKEKEPGNSALEEREQEKFNLKE ELERCKVQSSTLVSSLEAELSEVKIQTHIVQQENHLLKDELEKMKQLHRCPDLSDFQQKISS VLSYNEKLLKEKEALSEELNSCVDKLAKSSLLEHRIATMKQEQKSWEHQSASLKSQLVASQ EKVQNLEDTVQNVNLQMSRMKSDLRVTQQEKEALKQEVMSLHKQLQNAGGKSWAPEIAT HPSGLHNQQKRLSWDKLDHLMNEEQQLLWQENERQTMVQNTKAELTHSREKVRQLESN LLPKHQKHLNPSGTMNPTEQEKLSLKRECDQFQKEQSPANRKVSQMNSLEQELETIHLENE GLKKKQVKLDEQLMEMQHLRSTATPSPSPHAWDLQLLQQQACPMVPREQFLQLQRQLLQ AERINQHLQEELENRTSETNTPQGNQEQLVTVMEERMIEVEQKLKLVKRLLQEKVNQLKEQ LCKNTKADAMVKDLYVENAQLLKALEVTEQRQKTEAKKNYLLEEKIASLSNIVNLTPAPL TSTPPLRS (SEQ ID NO:6)

Example 5 Identification of PRAK/PN7098 Interaction

[0138] A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 786-1104 of PRAK (GB accession no. AF032437) as bait was performed. One clone that was identified by this procedure included novel protein fragment PN7098. The DNA sequence and the predicted protein sequence for PN7098 are set forth in Tables 36 and 37 respectively. TABLE 36 Nucleotide Sequence of PN7098 gccttggattttcaggttttcatcctgatacttgtttacttttctggggcagaaaagcttgcactaattgctctccatggtggctaatttttcaagagcttg attttaccttacattcataagctttgcaaaggaatgtttacaaagaaattgggaaatacaaacaaaaacaaagagtatcgtcagcagaaaaaggatc aagacttccccactgctggccagaccaaatcccccaaattttcttacacttttaaaagcactgtaaagaagattgcaaagtgttcatccactcacaact tatccactgaggaagacgaggccagtaaagagttttccctctcaccaacattcagttaccgagtagctattgccaatggcctacaaaagaatgctaa agtaaccaccagtgataatgaggatctgcttcaagagctctcttcaatcgagagttcctactcagaatcattaaatgaactaaggagtagcacagaa aaccaggcacaatcaacacacacaatgccagttagacgcaacagaaagagttcaagcagccttgcaccctctgagggcagctctgacgggga gcgtactctacatggcttaaaactgggagctttacgaaaactgagaaaatggaaaaaagagtcaagaatgtgtctcctcagactcagagttaagcac catgaaaaaatcctggggaataagaagtaagtctttggacagaactgtccgaaacccaaagacaaatgccctggagccagggttcagttcctctg gctgcattagccaaacacatgatgtcatggaaatgatctttaaggaacttcagggaataagtcagattgaaacagaactttctgaactacgagggca cgtcaatgctctcaagcactccatcgatgagatctccagcagtgtggaggttgtacaaagtgaaattgagcagttgcgcacagggtttgtccagtct cggagggaaactagagacatccatgattatattaagcacttaggtcatatgggtagcaaggcaagcctgagatttttaaatgtgactgaagaaagat ttgaatatgttgaaagcgtggtgtaccaaattctaatagataaaatgggtttttcagatgcaccaaatgctattaaaattgaatttgctcagaggatagg acaccagagagactgcccaaatgcaaagcctcgacccatacttgtgtactttgaaacccctcaacaaagggattctgtcttaaaaaagtcatataaa ctcaaaggaacaggcattggaatctcaacagatattctaactcatgacatcagagaaagaaaagagaaagggataccatcctcccagacatatga gagcatggctataaagttgtctactccagagccaaaaatcaagaagaacaattggcagtcacctgatgacagtgatgaagatcttgaatctgacct caatagaaacagttacgctgtgctttccaagtcagagcttctaacaaagggaagtacttccaagccaagctcaaaatcacacagtgctagatccaa gaataaaactgctaatagcagcagaatttcaaataaatcagattatgataaaatctcctcacagttgccagaatcagatatcttggaaaagcaaacca caacccattatgcagatgcaacacctctctggcactcacagagtgattttttcactgctaaacttagtcgttctgaatcagatttttccaaattgtgtcag tcttactcagaagatttttcagaaaatcagtttttcactagaactaatggaagctctctcctgtcatcttcggaccgggagctatggcagaggaaacag gaaggaacagcgaccctgtatgacagtcccaaggaccagcatttgaatggaagtgttcagggtatccaagggcagactgaaactgaaaacaca gaaactgtggatagtggaatgagtaatggcatggtgtgtgcatctggagaccggagtcattacagtgattctcagctctctttacatgaggatctttct ccatggaaggatggaatcaaggagctgatttaggcttggattcatccacccaggaaggttttgattatgaaacaaacagtctttttgaccaacagct tgatgtttacaataaagacctagaatacttgggaaagtgccacagtgatcttcaagatgactcagagagctacgacttaactcaagatgacaattctt ctccatgccctggcttggataatgaaccacaaggccagtgggttggccaatatgattcttatcagggagctaattctaatgagctataccaaaatca aaaccagttgtccatgatgtatcgaagtcaaagtgaattgcaaagtgatgattcagaggatgccccacccaaatcatggcatagtcgattaagcatt gacctttctgataagactttcagcttcccaaaatttggatctacactgcagagggctaaatcagccttggaagtagtatggaacaaaagcacacaga gtctgagtgggtatgaggacagtggctcttcattaatggggagatttcggacattatctcaatcaactgcaaatgagtcaagtaccacacttgactct gatgtctacacggagccctattactataaagcagaggatgaggaagattatactgaaccagtggctgacaatgaaacagattatgttgaagtcatg gaacaagtccttgctaaactagaaaacaggactagtattactgaaacagatgaacaaatgcaagcatatgatcacctttcatatgaaacaccttatga aaccccacaagatgagggttatgatggtccagcagatgatatggttagtgaagaggggttagaacccttaaatgaaacatcagctgagatggaaa taagagaagatgaaaaccaaaacattcctgaacagccagtggagatcacaaagccaaagagaattcgtccttctttcaaagaagcagctttaagg gcctataaaaagcaaatggcagagttggaagagaagatcttggctggagatagcagttctgtggatgaaaaggctcgaatagtaagtggcaatg atttggatgcttccaaattttctgcactccaggtgtgtggtggggctggaggtggactttatggtattgacagcatgccggatcttcgcagaaaaaaa actttgcctattgtccgagatgtggccatgaccctggctgcccggaaatctggactctccctggctatggtgattaggacatccctaaataatgagg aactgaaaatgcacgtcttcaagaagaccttgcaggcactgatctaccctatgtcttctaccatcccacacaattttgaggtctggacggctaccaca cccacctactgttatgagtgtgaagggctcctgtggggcattgcaaggcaaggcatgaagtgtctggagtgtggagtgaaatgccacgaaaagt gtcaggacctgctaaacgctgactgcttgcagagagcagcagaaaagagttctaaacatggtgccgaagacaagactcagaccattattacagc aatgaaagaaagaatgaagatcagggagaaaaaccggccagaagtatttgaagtaatccaggaaatgtttcagatttctaaagaagattttgtgca gtttacaaaggcggccaaacagagtgtactggatgggacatctaagtggtctgcaaaaataaccatcacagtggtttctgcacaagg SEQ ID NO:7)

[0139] TABLE 37 Predicted Amino Acid Seciuence of PN7098 MVANFFKSLILPYIHKLCKGMFTKKLGNTNKNKEYRQQKKDQDFPTAGQTKSPKFSYTFKS TVKKIAKCSSTHNLSTEEDEASKEFSLSPTFSYRVAIANGLQKNAKVTTSDNEDLLQELSSIE SSYSESLNELRSSTENQAQSTHTMPVRRNRKSSSSLAPSEGSSDGERTLHGLKLGALRKLRK WKKSQECVSSDSELSTMKKSWGIRSKSLDRTVRNPKTNALEPGFSSSGCISQTHDVMEMIF KELQGISQIETELSELRGHVNALKHSIDEISSSVEVVQSEIEQLRTGFVQSRRETRDIHDYIKH LGHMGSKASLRFLNVTEERFEYVESVVYQILIDKMGFSDAPNAIKIEFAQRIGHQRDCPNAK PRPILVYFETPQQRDSVLKKSYKLKGTGIGISTDILTHDIRERKEKGIPSSQTYESMAIKLSTPE PKIKKNNWQSPDDSDEDLESDLNRNSYAVLSKSELLTKGSTSKPSSKSHSARSKNKTANSSR ISNKSDYDKISSQLPESDILEKQTTTHYADATPLWHSQSDFFTAKLSRSESDFSKCQSYSED FSENQFFTRTNGSSLLSSSDRELWQRKQEGTATLYDSPKDQHLNGSVQGIQGQTETENTETV DSGMSNGMVCASGDRSHYSDSQLSLHEDLSPWKEWNQGADLGLDSSTQEGFDYETNSLFD QQLDVYNKDLEYLGKCHSDLQDDSESYDLTQDDNSSPCPGLDNEPQGQWVGQYDSYQGA NSNELYQNQNQLSMMYRSQSELQSDDSEDAPPKSWHSRLSIDLSDKTFSFPKFGSTLQRAK SALEVVWNKSTQSLSGYEDSGSSLMGRFRTLSQSTANESSTTLDSDVYTEPYYYKAEDEED YTEPVADNETDYVEVMEQVLAKLENRTSITETDEQMQAYDHLSYETPYETPQDEGYDGPA DDMVSEEGLEPLNETSAEMEIREDENQNIPEQPVEITKPKRIRPSFKEAALRAYKKQMAELE EKILAGDSSSVDEKARIVSGNDLDASKFSALQVCGGAGGGLYGIDSMPDLRRKKTLPIVRD VAMTLAARKSGLSLAMVIRTSLNNEELKMHVFKKTLQALIYPMSSTIPHNFEVWTATTPTY CYECEGLLWGIARQGMKCLECGVKCHEKCQDLLNADCLQRAAEKSSKHGAEDKTQTIITA MKERMKIREKNRPEVFEVIQEMFQISKEDFVQFTKAAKQSVLDGTSKWSAKITITVVSAQX (SEQ ID NO:8)

EXAMPLES 6-32 Identification of Protein-Protein Interactions

[0140] A yeast two-hybrid system as described in Example 1 using amino acids of the bait as set forth in Table 38 was performed. The clone that was identified by this procedure for each bait is set forth in Table 38 as the prey. The “AA” refers to the amino acids of the bait or prey. The “NUC” refers to the nucleotides of the bait or prey. The Accession numbers refer to GB: GenBank and SP: Swiss Protein accession numbers. TABLE 38 Ex. BAIT ACCESSION COORDINATES PREY ACCESSION COORDINATES 6 p38 ALPHA SP: Q13083 AA 1-130 JNK3 ALPHA2 SP: P53779 AA 371-464 7 p38 ALPHA SP: Q13083 AA 1-130 C-NAP1 GB: AF049105 AA 1362-1579 8 p38 ALPHA SP: Q13083 AA 194-319 VINCULIN SP: P18206 AA 933-1067 9 p38 ALPHA (K53M MUTANT) SP: Q13083 AA 1-361 SPLICING FACTOR PSF SP: P23246 AA 282-577 10 MAPKAP-K2 SP: P49137 AA 238-325 LEUCINE-RICH PROTEIN L130 SP: P42704 AA 31-263 11 MAPKAP-K2 SP: P49137 AA 134-325 cAMP-DEP PROTEIN KINASE SP: P10644 AA 20-382 12 MAPKAP-K2 SP: P49137 AA 134-325 SET SP: Q01105 AA 106-239 13 MAPKAP-K2 SP: P49137 AA 1-338 TL21 GB: X75692 NUC 6-276 14 MAPKAP-K2 (K93M, SP: P49137 AA 1-338 ERK3 SP: Q16659 AA 19-509 T222D,T334D) 15 MAPKAP-K3 GB: U09578 AA 1-304 THROMBOSPONDIN 3 SP: P49746 AA 215-366 16 MAPKAP-K3 GB: U09578 AA 114-304 MALATE DEHYDROGENASE SP: P40925 AA 1-326 17 MAPKAP-K3 GB: U09578 AA 114-304 GA17 GB: AF064603 AA 1-363 18 MAPKAP-K3 GB: U09578 AA 114-304 CALPAIN 4 SMALL SUBUNIT SP: P04632 AA 102-269 19 MAPKAP-K3 GB: U09578 AA 217-304 BAT3 SP: P46379 AA 190-473 20 MSK-1 GB: AF074393 AA 426-686 ABLIM GB: AF005654 AA 197-340 21 MSK-1 GB: AF074393 AA 426-686 KIAA0144 SP: Q14157 AA 690-857 22 PRAK GB: AF032437 AA 3-304 KENDRIN GB: U52962 AA 191-574 23 PRAK GB: AF032437 AA 3-304 HOMEOTIC PROTEIN PROX1 SP: Q92786 AA 203-464 24 PRAK GB: AF032437 AA 3-304 HOOK1 GB: AA 1-413 AF0414923 25 PRAK GB: AF032437 AA 3-304 IG HEAVY CHAIN CONSTANT SP: P01857 AA 105-192 REGION 26 PRAK GB: AF032437 AA 3-304 GOLGIN-95 SP: Q08379 AA 22-482 27 PRAK GB: AF032437 AA 3-304 KIAA0555 GB: AB011127 AA 461-583 28 PRAK GB: AF032437 AA 198-304 LEUCINE-RICH PROTEIN L130 SP: P42704 AA 31-263 29 PRAK GB: AF032437 AA 304-471 ERK3 SP: Q16659 AA 36-502 30 PRAK GB: AF032437 AA 304-471 cAMP-DEP PROTEIN KINASE SP: P10644 AA 19-141 31 PRAK GB: AF032437 AA 3-304 AL117538 GB: AL117538 AA 1-43 32 PRAK GB: AF032437 AA 3-304 AL117237 GB: AL117237 AA 401-488

Example 33 Generation of Polyclonal Antibody Against Protein Complexes

[0141] As shown above, p38 alpha interacts with CYT4 to form a complex. A complex of the two proteins is prepared, e.g., by mixing purified preparations of each of the two proteins. If desired, the protein complex can be stabilized by cross-linking the proteins in the complex, by methods known to those of skill in the art. The protein complex is used to immunize rabbits and mice using a procedure similar to that described by Harlow et al. (1988). This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al., 1993).

[0142] Briefly, purified protein complex is used as immunogen in rabbits. Rabbits are immunized with 100 μg of the protein in complete Freund's adjuvant and boosted twice in three-week intervals, first with 100 μg of immunogen in incomplete Freund's adjuvant, and followed by 100 μg of immunogen in PBS. Antibody-containing serum is collected two weeks thereafter. The antisera is preadsorbed with P38 alpha and CYT4, such that the remaining antisera comprises antibodies which bind conformational epitopes, i.e., complex-specific epitopes, present on the P38 alpha-CYT4 complex but not on the monomers.

[0143] Polyclonal antibodies against each of the complexes set forth in Tables 1-31 are prepared in a similar manner by mixing the specified proteins together, immunizing an animal and isolating antibodies specific for the protein complex, but not for the individual proteins.

[0144] Polyclonal antibodies against each of the proteins set forth in Tables 33, 35 and 37 are prepared in a similar manner by immunizing an animal with the protein and isolating antibodies specific for the protein.

Example 34 Generation of Monoclonal Antibodies Specific for Protein Complexes

[0145] Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising P38 alpha/CYT4 complexes conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known in the art. The complexes can be prepared as described in Example 33, and may also be stabilized by cross-linking. The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 μg of immunogen, and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.

[0146] Spleens are removed from immune mice and a single-cell suspension is prepared (Harlow et al., 1988). Cell fusions are performed essentially as described by Kohler et al. (1975). Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, Md.) or NS-1 myeloma cells are fused with immune spleen cells using polyethylene glycol as described by Harlow et al. (1988). Cells are plated at a density of 2×10⁵ cells/well in 96-well tissue culture plates. Individual wells are examined for growth, and the supernatants of wells with growth are tested for the presence of P38 alpha/CYT4 complex-specific antibodies by ELISA or RIA using P38 alpha/CYT4 complex as target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.

[0147] Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibodies for characterization and assay development. Antibodies are tested for binding to P38 alpha alone or to CYT4 alone, to determine which are specific for the P38 alpha/CYT4 complex as opposed to those that bind to the individual proteins.

[0148] Monoclonal antibodies against each of the complexes set forth in Tables 1-31 are prepared in a similar manner by mixing the specified proteins together, immunizing an animal, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein complex, but not for the individual proteins.

[0149] Monoclonal antibodies against each of the proteins set forth in Tables 33, 35 and 37 are prepared in a similar manner by immunizing an animal with the protein, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein.

Example 35 In vitro Identification of Modulators for Protein-Protein Interactions

[0150] The present invention is useful in screening for agents that modulate the interaction of P38 alpha and CYT4. The knowledge that P38 alpha and CYT4 form a complex is useful in designing such assays. Candidate agents are screened by mixing P38 alpha and CYT4 (a) in the presence of a candidate agent, and (b) in the absence of the candidate agent. The amount of complex formed is measured for each sample. An agent modulates the interaction of P38 alpha and CYT4 if the amount of complex formed in the presence of the agent is greater than (promoting the interaction), or less than (inhibiting the interaction) the amount of complex formed in the absence of the agent. The amount of complex is measured by a binding assay, which shows the formation of the complex, or by using antibodies immunoreactive to the complex.

[0151] Briefly, a binding assay is performed in which immobilized P38 alpha is used to bind labeled CYT4. The labeled CYT4 is contacted with the immobilized P38 alpha under aqueous conditions that permit specific binding of the two proteins to form a P38 alpha/CYT4 complex in the absence of an added test agent. Particular aqueous conditions may be selected according to conventional methods. Any reaction condition can be used as long as specific binding of P38 alpha/CYT4 occurs in the control reaction. A parallel binding assay is performed in which the test agent is added to the reaction mixture. The amount of labeled CYT4 bound to the immobilized P38 alpha is determined for the reactions in the absence or presence of the test agent. If the amount of bound, labeled CYT4 in the presence of the test agent is different than the amount of bound labeled CYT4 in the absence of the test agent, the test agent is a modulator of the interaction of P38 alpha and CYT4.

[0152] Candidate agents for modulating the interaction of each of the protein complexes set forth in Tables 1-31 are screened in vitro in a similar manner.

Example 36 In vivo Identification of Modulators for Protein-Protein Interactions

[0153] In addition to the in vitro method described in Example 35, an in vivo assay can also be used to screen for agents which modulate the interaction of P38 alpha and CYT4. Briefly, a yeast two-hybrid system is used in which the yeast cells express (1) a first fusion protein comprising P38 alpha or a fragment thereof and a first transcriptional regulatory protein sequence, e.g., GAL4 activation domain, (2) a second fusion protein comprising CYT4 or a fragment thereof and a second transcriptional regulatory protein sequence, e.g., GAL4 DNA-binding domain, and (3) a reporter gene, e.g., β-galactosidase, which is transcribed when an intermolecular complex comprising the first fusion protein and the second fusion protein is formed. Parallel reactions are performed in the absence of a test agent as the control and in the presence of the test agent. A functional P38 alpha/CYT4 complex is detected by detecting the amount of reporter gene expressed. If the amount of reporter gene expression in the presence of the test agent is different than the amount of reporter gene expression in the absence of the test agent, the test agent is a modulator of the interaction of P38 alpha and CYT4.

[0154] Candidate agents for modulating the interaction of each of the protein complexes set forth in Tables 1-31 are screened in vivo in a similar manner.

[0155] While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.

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[0212] PCT Published Application No. WO 97/27296

[0213] PCT Published Application No. WO 99/65939

[0214] U.S. Pat. No. 5,622,852

[0215] U.S. Pat. No. 5,773,218

1 8 1 40 DNA Primer for yeast two-hybrid assays 1 gcaggaaaca gctatgacca tacagtcagc ggccgccacc 40 2 39 DNA Primer for yeast two-hybrid assays 2 acggccagtc gcgtggagtg ttatgtcatg cggccgcta 39 3 5153 DNA Homo sapiens CDS (141)..(2159) 3 gccgcgtcga cgtcgaccca gactggagcg acgtttaaag aaggggcaga atcgctgggg 60 agtgcggctt cttcttgttg ggggactccc agccttccgc gcgtccggag gaggagaagc 120 ggcggcgccg ggaagcaggc atg gag agt aga aaa ctg att tct gct aca gac 173 Met Glu Ser Arg Lys Leu Ile Ser Ala Thr Asp 1 5 10 att cag tac tct ggc agt ctg ctg aac tcc ttg aat gag caa cgt ggc 221 Ile Gln Tyr Ser Gly Ser Leu Leu Asn Ser Leu Asn Glu Gln Arg Gly 15 20 25 cat gga ctc ttc tgt gat gtt acc gtt att gtg gaa gac cga aaa ttc 269 His Gly Leu Phe Cys Asp Val Thr Val Ile Val Glu Asp Arg Lys Phe 30 35 40 cgg gct cac aag aat att ctt tca gct tct agt acc tac ttc cat cag 317 Arg Ala His Lys Asn Ile Leu Ser Ala Ser Ser Thr Tyr Phe His Gln 45 50 55 ctc ttc tct gtt gct ggg caa gtt gtt gaa ctg agc ttt ata aga gca 365 Leu Phe Ser Val Ala Gly Gln Val Val Glu Leu Ser Phe Ile Arg Ala 60 65 70 75 gag atc ttt gca gaa att ctc aat tat atc tat agt tct aaa att gtt 413 Glu Ile Phe Ala Glu Ile Leu Asn Tyr Ile Tyr Ser Ser Lys Ile Val 80 85 90 cgt gtt aga tca gat ttg ctt gat gag tta att aaa tca ggg cag tta 461 Arg Val Arg Ser Asp Leu Leu Asp Glu Leu Ile Lys Ser Gly Gln Leu 95 100 105 tta gga gtg aaa ttt ata gca gag ctt ggt gtc cca ttg tca cag gtt 509 Leu Gly Val Lys Phe Ile Ala Glu Leu Gly Val Pro Leu Ser Gln Val 110 115 120 aaa agc atc tca ggt aca gcg cag gat ggt aat act gag cct tta cct 557 Lys Ser Ile Ser Gly Thr Ala Gln Asp Gly Asn Thr Glu Pro Leu Pro 125 130 135 cct gat tct ggt gac aag aac ctt gta ata cag aaa tca aaa gat gaa 605 Pro Asp Ser Gly Asp Lys Asn Leu Val Ile Gln Lys Ser Lys Asp Glu 140 145 150 155 gcc caa gat aat ggg gct act ata atg cct att ata aca gag tct ttt 653 Ala Gln Asp Asn Gly Ala Thr Ile Met Pro Ile Ile Thr Glu Ser Phe 160 165 170 tca tta tct gcc gaa gat tat gaa atg aaa aag atc att gtt acc gat 701 Ser Leu Ser Ala Glu Asp Tyr Glu Met Lys Lys Ile Ile Val Thr Asp 175 180 185 tct gat gat gat gat gat gat gat gtc att ttt tgc tcc gag att ctg 749 Ser Asp Asp Asp Asp Asp Asp Asp Val Ile Phe Cys Ser Glu Ile Leu 190 195 200 ccc aca aag gag act ttg ccg agt aat aac aca gtg gca cag gtc caa 797 Pro Thr Lys Glu Thr Leu Pro Ser Asn Asn Thr Val Ala Gln Val Gln 205 210 215 tct aac cca ggc cct gtt gct att tca gat gtt gca cct agt gct agc 845 Ser Asn Pro Gly Pro Val Ala Ile Ser Asp Val Ala Pro Ser Ala Ser 220 225 230 235 aat aac tcg ccc cct tta aca aat atc aca cct act cag aaa ctt cct 893 Asn Asn Ser Pro Pro Leu Thr Asn Ile Thr Pro Thr Gln Lys Leu Pro 240 245 250 act cct gtg aat cag gca act ttg agc caa aca caa gga agt gaa aaa 941 Thr Pro Val Asn Gln Ala Thr Leu Ser Gln Thr Gln Gly Ser Glu Lys 255 260 265 ttg ttg gta tct tca gct cca aca cat ctg act ccc aat att att ttg 989 Leu Leu Val Ser Ser Ala Pro Thr His Leu Thr Pro Asn Ile Ile Leu 270 275 280 tta aat cag aca cca ctt tct aca cca cca aat gtc agt tct tca ctt 1037 Leu Asn Gln Thr Pro Leu Ser Thr Pro Pro Asn Val Ser Ser Ser Leu 285 290 295 cca aat cat atg ccc tct tca atc aat tta ctt gtg cag aat cag cag 1085 Pro Asn His Met Pro Ser Ser Ile Asn Leu Leu Val Gln Asn Gln Gln 300 305 310 315 aca cca aac agt gct att tta aca gga aac aag gcc aat gaa gag gag 1133 Thr Pro Asn Ser Ala Ile Leu Thr Gly Asn Lys Ala Asn Glu Glu Glu 320 325 330 gag gag gaa ata ata gat gat gat gat gac act att agc tcc agt cct 1181 Glu Glu Glu Ile Ile Asp Asp Asp Asp Asp Thr Ile Ser Ser Ser Pro 335 340 345 gac tcg gcc gtc agt aat aca tct ttg gtc cca cag gct gat acc tcc 1229 Asp Ser Ala Val Ser Asn Thr Ser Leu Val Pro Gln Ala Asp Thr Ser 350 355 360 caa aat acc agt ttt gat gga tca tta ata cag aag atg cag att cct 1277 Gln Asn Thr Ser Phe Asp Gly Ser Leu Ile Gln Lys Met Gln Ile Pro 365 370 375 aca ctt ctt caa gaa cca ctt tcc aat tcc tta aaa att tca gat ata 1325 Thr Leu Leu Gln Glu Pro Leu Ser Asn Ser Leu Lys Ile Ser Asp Ile 380 385 390 395 att act aga aat act aat gat cca ggc gta gga tca aaa cat cta atg 1373 Ile Thr Arg Asn Thr Asn Asp Pro Gly Val Gly Ser Lys His Leu Met 400 405 410 gag ggt cag aag atc att act tta gat aca gct act gaa att gaa ggc 1421 Glu Gly Gln Lys Ile Ile Thr Leu Asp Thr Ala Thr Glu Ile Glu Gly 415 420 425 tta tcg act ggt tgc aag gtt tat gca aat atc ggt gaa gat act tat 1469 Leu Ser Thr Gly Cys Lys Val Tyr Ala Asn Ile Gly Glu Asp Thr Tyr 430 435 440 gat ata gtg atc cct gtc aaa gat gac cct gat gaa ggg gag gcc aga 1517 Asp Ile Val Ile Pro Val Lys Asp Asp Pro Asp Glu Gly Glu Ala Arg 445 450 455 ctt gag aat gaa ata cca aaa acg tct ggc agc gag atg gca aac aaa 1565 Leu Glu Asn Glu Ile Pro Lys Thr Ser Gly Ser Glu Met Ala Asn Lys 460 465 470 475 cgt atg aaa gta aaa cat gat gat cac tat gag tta ata gta gat gga 1613 Arg Met Lys Val Lys His Asp Asp His Tyr Glu Leu Ile Val Asp Gly 480 485 490 agg gtc tat tat atc tgt att gta tgc aaa agg tca tat gtc tgt ctg 1661 Arg Val Tyr Tyr Ile Cys Ile Val Cys Lys Arg Ser Tyr Val Cys Leu 495 500 505 aca agc ttg cgg aga cat ttt aac att cat tct tgg gag aag aag tat 1709 Thr Ser Leu Arg Arg His Phe Asn Ile His Ser Trp Glu Lys Lys Tyr 510 515 520 ccg tgc cgt tac tgt gag aag gta ttt cct ctt gca gaa tat cgc aca 1757 Pro Cys Arg Tyr Cys Glu Lys Val Phe Pro Leu Ala Glu Tyr Arg Thr 525 530 535 aag cat gaa att cat cac aca ggg gag cga agg tat cag tgt ttg gcc 1805 Lys His Glu Ile His His Thr Gly Glu Arg Arg Tyr Gln Cys Leu Ala 540 545 550 555 tgt ggc aaa tct ttc atc aac tat cag ttt atg tct tca cat ata aag 1853 Cys Gly Lys Ser Phe Ile Asn Tyr Gln Phe Met Ser Ser His Ile Lys 560 565 570 tca gtt cat agt caa gat cct tct ggg gac tca aag ctt tat cgt tta 1901 Ser Val His Ser Gln Asp Pro Ser Gly Asp Ser Lys Leu Tyr Arg Leu 575 580 585 cat cca tgc agg tct tta caa atc aga caa tat gca tat ctt tcc gat 1949 His Pro Cys Arg Ser Leu Gln Ile Arg Gln Tyr Ala Tyr Leu Ser Asp 590 595 600 aga tca agc act att cct gca atg aag gat gat ggt att ggg tat aag 1997 Arg Ser Ser Thr Ile Pro Ala Met Lys Asp Asp Gly Ile Gly Tyr Lys 605 610 615 gtt gac act gga aaa gaa cct cca gta ggg acc act aca tct act cag 2045 Val Asp Thr Gly Lys Glu Pro Pro Val Gly Thr Thr Thr Ser Thr Gln 620 625 630 635 aac aag cca atg acc tgg gaa gat att ttt att cag cag gaa aat gat 2093 Asn Lys Pro Met Thr Trp Glu Asp Ile Phe Ile Gln Gln Glu Asn Asp 640 645 650 tca att ttt aaa caa aat gta aca gat ggc agt act gag ttt gaa ttt 2141 Ser Ile Phe Lys Gln Asn Val Thr Asp Gly Ser Thr Glu Phe Glu Phe 655 660 665 ata ata cca gag tct tac taaactcctt tgaaatacta gaaagttttg 2189 Ile Ile Pro Glu Ser Tyr 670 ttttggatga tggggcaggg gtttcagaag atctgtaaaa caaattaagg tgcgaacaag 2249 ttaatttgat ctgccacatt atctgaagga agtgtagtgg gatttttgtt gataattttt 2309 agaagcaaat tttcctgaaa gttttgagta gaggtgagac cccctcccca agtatctgtt 2369 tatatagtta gttttcagct catttaaaag aggcaaaaat taaaagcttg gagagatagt 2429 ttcctgaata gaatttgaag cagtctgaat gttctttgaa aataactgga gttattagca 2489 taccctagta catcttacag ctttcccctt ccatgttagc actttactgc tgaattctca 2549 attttcttaa cattgagaca ataaatgtgt gttttgtctt gtatatggca taaagagtaa 2609 ataagtttta gagttgttct ggaaaatgtc agaataagtc agtacttggg ttgtgtaatc 2669 tgctagtcca agcgaacagc aacctcctgc taccctccct ctatgaaaat agccatgcag 2729 acaagtctct catctgaaga acaaattaga tttagctaat tagaattaat cctggctttc 2789 attgccatag tctgtaaaag actttggtgg ctagaccact ttataccttt gcagtgtggt 2849 ctctgggggc aaaaaactaa tgaaaacaat ctctgtaatg gcagatagga ggagatgaaa 2909 agttctgttg catggatttt taattctctg gctaccacat agtagagaat ggaatgaaga 2969 tttccttttg gcttcttaag gttaaaaata ttcccatgaa catgaaaatt ttcaaatttt 3029 gaatctgaaa gccaccaaat gtatctttat gtataaatcc ttgtaaatga tagattccat 3089 gggtgagact ttacatattt tgggtgggag gctactggca tatattttta aatgttcata 3149 ttgcgtagaa tctccactag gaagtcttta tttgaaatag ttgaatcagt gatctagtat 3209 tttcctttcg gcaagatttg ttaggttttt accccttcta aaataagttt tattccatct 3269 gcaaattgct gcaatattat agtaatcaga aactacataa ggaatgttat ataggcttgt 3329 cagttcccgt ttttcttgac aacaataaat accactttta aaaatgacac atatttaaac 3389 acttagaaaa taaagttaac acttactgaa gtgctagtac taaactgtgc tagtactaaa 3449 agaaaacagg ttggaacata catatagcct agcatttata acagaattgt tgaacgtctg 3509 taaatgattt tttttttttt tgcaaaggaa aaaattgata ctggaaaaga ttgttgtgca 3569 tagttattag tcatttgtaa ccttgcttaa gtatttctta gtccaacata gatattttct 3629 ttctcctgac catgtatttt aaaatatagt ctatttcttg actttgaact taaagcttta 3689 atcataattt ctcatgtata catcgttctt ctgatggtaa gctggatttg aaggtagtgg 3749 tttcagtgtt tcttaagttg gtagctgagg gtatcaggca tcagttcatg caataataca 3809 agaaaaaaaa tcctttgctt gccaagaggt agagtgatgt gcatttatct gttttctgtt 3869 ctgtaagtct agaccttcaa accatttgta aactaacccc tgggaaattt gaaattacct 3929 gataacttaa gactctgtga tctctggaat caccatatgt ttcttttttg tgtagatatt 3989 aataacatta ctctttgact atagtgtgca ctctgaaatg tactcagtga aaatttgttt 4049 tgagtttcat taatgctatt tcaccagtta gacataatta cttctaccga tgtgaatgat 4109 acggatgccg gcagagcttc cagatctttc agactcaact gctaggtcaa ttagtttgtc 4169 ataataaaac ttggcagatt ctacaagtct attatgacaa accaggaact aattctataa 4229 tggaaaacta tccattctga ataataggta tgtaattatt tgctgctgct gctgtgctct 4289 gtaaattctg aatatgacat ttaaactctg tgcctactaa aggtatcttc tggagttttt 4349 gggaggagag aaactggaaa attaaattgt atttttgcca gaagactctt acttgcatgt 4409 gtctcagggt cttcagtttt tctataagtt tccatatcca aagttcagaa ttcatgtgaa 4469 atacttcttt ggggcaaaag tccttcattc ctggtattta ttggattgga aatctgtagc 4529 aagatgctgt ttaaaattac catattgttt ttttatctta tacttagctc tctggctatt 4589 gaacttcctt ttcttgtttg aagttagctt caaatttgct cctatgctaa attacctgta 4649 aatattctgg ataggaacta cttgaaatag taatttgtta aaagatatga caaaatgaaa 4709 atgcttaaac tacagaaatt taaaaatgcc ataacaatct tgcaagacta actttaaaat 4769 atactttaaa tgattattat gattttggtg gtaacgatcc cccacacaca accactatga 4829 agaaataatg ccgcattttt cccccattgt accaaaaaga taaaaaaatg gtaaacactg 4889 atcaaggtat tttgtattgt caaggcatgc atattctaaa gaattaaatg ctaacttaac 4949 agcactggct ttctggctgg tcaactatat gaaaccttgt tcattcctcc gagtactgta 5009 atgttcacac ttgtacaatc ttccctgtca tgactttaag ttctactttt cattaaccat 5069 ggcctgatat tagttcttag agcttcttgt ggcaaaaata aaatgattta attctgaaaa 5129 aaaaaaaaaa aaaaaaaaaa aaaa 5153 4 673 PRT Homo sapiens 4 Met Glu Ser Arg Lys Leu Ile Ser Ala Thr Asp Ile Gln Tyr Ser Gly 1 5 10 15 Ser Leu Leu Asn Ser Leu Asn Glu Gln Arg Gly His Gly Leu Phe Cys 20 25 30 Asp Val Thr Val Ile Val Glu Asp Arg Lys Phe Arg Ala His Lys Asn 35 40 45 Ile Leu Ser Ala Ser Ser Thr Tyr Phe His Gln Leu Phe Ser Val Ala 50 55 60 Gly Gln Val Val Glu Leu Ser Phe Ile Arg Ala Glu Ile Phe Ala Glu 65 70 75 80 Ile Leu Asn Tyr Ile Tyr Ser Ser Lys Ile Val Arg Val Arg Ser Asp 85 90 95 Leu Leu Asp Glu Leu Ile Lys Ser Gly Gln Leu Leu Gly Val Lys Phe 100 105 110 Ile Ala Glu Leu Gly Val Pro Leu Ser Gln Val Lys Ser Ile Ser Gly 115 120 125 Thr Ala Gln Asp Gly Asn Thr Glu Pro Leu Pro Pro Asp Ser Gly Asp 130 135 140 Lys Asn Leu Val Ile Gln Lys Ser Lys Asp Glu Ala Gln Asp Asn Gly 145 150 155 160 Ala Thr Ile Met Pro Ile Ile Thr Glu Ser Phe Ser Leu Ser Ala Glu 165 170 175 Asp Tyr Glu Met Lys Lys Ile Ile Val Thr Asp Ser Asp Asp Asp Asp 180 185 190 Asp Asp Asp Val Ile Phe Cys Ser Glu Ile Leu Pro Thr Lys Glu Thr 195 200 205 Leu Pro Ser Asn Asn Thr Val Ala Gln Val Gln Ser Asn Pro Gly Pro 210 215 220 Val Ala Ile Ser Asp Val Ala Pro Ser Ala Ser Asn Asn Ser Pro Pro 225 230 235 240 Leu Thr Asn Ile Thr Pro Thr Gln Lys Leu Pro Thr Pro Val Asn Gln 245 250 255 Ala Thr Leu Ser Gln Thr Gln Gly Ser Glu Lys Leu Leu Val Ser Ser 260 265 270 Ala Pro Thr His Leu Thr Pro Asn Ile Ile Leu Leu Asn Gln Thr Pro 275 280 285 Leu Ser Thr Pro Pro Asn Val Ser Ser Ser Leu Pro Asn His Met Pro 290 295 300 Ser Ser Ile Asn Leu Leu Val Gln Asn Gln Gln Thr Pro Asn Ser Ala 305 310 315 320 Ile Leu Thr Gly Asn Lys Ala Asn Glu Glu Glu Glu Glu Glu Ile Ile 325 330 335 Asp Asp Asp Asp Asp Thr Ile Ser Ser Ser Pro Asp Ser Ala Val Ser 340 345 350 Asn Thr Ser Leu Val Pro Gln Ala Asp Thr Ser Gln Asn Thr Ser Phe 355 360 365 Asp Gly Ser Leu Ile Gln Lys Met Gln Ile Pro Thr Leu Leu Gln Glu 370 375 380 Pro Leu Ser Asn Ser Leu Lys Ile Ser Asp Ile Ile Thr Arg Asn Thr 385 390 395 400 Asn Asp Pro Gly Val Gly Ser Lys His Leu Met Glu Gly Gln Lys Ile 405 410 415 Ile Thr Leu Asp Thr Ala Thr Glu Ile Glu Gly Leu Ser Thr Gly Cys 420 425 430 Lys Val Tyr Ala Asn Ile Gly Glu Asp Thr Tyr Asp Ile Val Ile Pro 435 440 445 Val Lys Asp Asp Pro Asp Glu Gly Glu Ala Arg Leu Glu Asn Glu Ile 450 455 460 Pro Lys Thr Ser Gly Ser Glu Met Ala Asn Lys Arg Met Lys Val Lys 465 470 475 480 His Asp Asp His Tyr Glu Leu Ile Val Asp Gly Arg Val Tyr Tyr Ile 485 490 495 Cys Ile Val Cys Lys Arg Ser Tyr Val Cys Leu Thr Ser Leu Arg Arg 500 505 510 His Phe Asn Ile His Ser Trp Glu Lys Lys Tyr Pro Cys Arg Tyr Cys 515 520 525 Glu Lys Val Phe Pro Leu Ala Glu Tyr Arg Thr Lys His Glu Ile His 530 535 540 His Thr Gly Glu Arg Arg Tyr Gln Cys Leu Ala Cys Gly Lys Ser Phe 545 550 555 560 Ile Asn Tyr Gln Phe Met Ser Ser His Ile Lys Ser Val His Ser Gln 565 570 575 Asp Pro Ser Gly Asp Ser Lys Leu Tyr Arg Leu His Pro Cys Arg Ser 580 585 590 Leu Gln Ile Arg Gln Tyr Ala Tyr Leu Ser Asp Arg Ser Ser Thr Ile 595 600 605 Pro Ala Met Lys Asp Asp Gly Ile Gly Tyr Lys Val Asp Thr Gly Lys 610 615 620 Glu Pro Pro Val Gly Thr Thr Thr Ser Thr Gln Asn Lys Pro Met Thr 625 630 635 640 Trp Glu Asp Ile Phe Ile Gln Gln Glu Asn Asp Ser Ile Phe Lys Gln 645 650 655 Asn Val Thr Asp Gly Ser Thr Glu Phe Glu Phe Ile Ile Pro Glu Ser 660 665 670 Tyr 5 10625 DNA Homo sapiens CDS (544)..(6960) 5 cttattttga aaacatttac atagtgatta gttaacccaa cagaccaatc ctgggaagac 60 agccagagcc tgcagcacct tagtaacaga aaaactgata attaggagaa gagacctgtc 120 caagaccagg aacctggacc aaaattgtgc catgttgctt tactttaatg agtggcccca 180 gtaaaaactg agctgtatgg cagagctgtt cacatttatc ttctgtgtcc acccagttct 240 gctgaaaccc ctggcaagat cgtggccctg ttgtagcttg tcatgttttg aacagctgtc 300 tatggaaaga aagcaaacac aacctagagc aacattgatt tgttttagaa agctctttta 360 ttttcagttc tggctgtgtt caacatctta gcttacgttt ttcatgttgt aatgatctgc 420 cgtatggacg atcacctcta agttagagag ttctgtaatt tggcttggat taaagatgct 480 tggttagtga aagctgctgc tttttttata gtcaaaggac tggttctgag agccttgttg 540 cag atg gct gag gtc acc gtc cca agg gtg tat gtc gtg ttt ggc atc 588 Met Ala Glu Val Thr Val Pro Arg Val Tyr Val Val Phe Gly Ile 1 5 10 15 cat tgc atc atg gcg aag gca tct tca gat gtg cag gtt tca ggc ttt 636 His Cys Ile Met Ala Lys Ala Ser Ser Asp Val Gln Val Ser Gly Phe 20 25 30 cat cgg aaa atc cag cac gtt aaa aat gaa ctt tgc cac atg ttg agc 684 His Arg Lys Ile Gln His Val Lys Asn Glu Leu Cys His Met Leu Ser 35 40 45 ttg gag gag gtg gcc cca gtg ctg cag cag aca tta ctt cag gac aac 732 Leu Glu Glu Val Ala Pro Val Leu Gln Gln Thr Leu Leu Gln Asp Asn 50 55 60 ctc ttg ggc agg gta cat ttt gac caa ttt aaa gaa gca tta ata ctc 780 Leu Leu Gly Arg Val His Phe Asp Gln Phe Lys Glu Ala Leu Ile Leu 65 70 75 atc ttg tcc aga act ctg tca aat gaa gaa cac ttt caa gaa cca gac 828 Ile Leu Ser Arg Thr Leu Ser Asn Glu Glu His Phe Gln Glu Pro Asp 80 85 90 95 tgc tca cta gaa gct cag ccc aaa tat gtt aga ggt ggg aag cgt tac 876 Cys Ser Leu Glu Ala Gln Pro Lys Tyr Val Arg Gly Gly Lys Arg Tyr 100 105 110 gga cga agg tcc ttg ccc gag ttc caa gag tcc gtg gag gag ttt cct 924 Gly Arg Arg Ser Leu Pro Glu Phe Gln Glu Ser Val Glu Glu Phe Pro 115 120 125 gaa gtg acg gtg att gag cca ctg gat gaa gaa gcg cgg cct tca cac 972 Glu Val Thr Val Ile Glu Pro Leu Asp Glu Glu Ala Arg Pro Ser His 130 135 140 atc cca gcc ggt gac tgc agt gag cac tgg aag acg caa cgc agt gag 1020 Ile Pro Ala Gly Asp Cys Ser Glu His Trp Lys Thr Gln Arg Ser Glu 145 150 155 gag tat gaa gcg gaa ggc cag tta agg ttt tgg aac cca gat gac ttg 1068 Glu Tyr Glu Ala Glu Gly Gln Leu Arg Phe Trp Asn Pro Asp Asp Leu 160 165 170 175 aat gct tca cag agt gga tct tcc cct ccc caa gac tgg ata gaa gag 1116 Asn Ala Ser Gln Ser Gly Ser Ser Pro Pro Gln Asp Trp Ile Glu Glu 180 185 190 aaa ctg caa gaa gtt tgt gaa gat ttg ggg atc acc cgt gat ggt cac 1164 Lys Leu Gln Glu Val Cys Glu Asp Leu Gly Ile Thr Arg Asp Gly His 195 200 205 ctg aac cgg aag aag ctg gtc tcc atc tgt gag cag tat ggt tta cag 1212 Leu Asn Arg Lys Lys Leu Val Ser Ile Cys Glu Gln Tyr Gly Leu Gln 210 215 220 aat gtg gat gga gag atg ctc gag gaa gta ttc cat aat ctt gat cct 1260 Asn Val Asp Gly Glu Met Leu Glu Glu Val Phe His Asn Leu Asp Pro 225 230 235 gac ggt aca atg agt gta gaa gat ttt ttc tat ggt ttg ttt aaa aat 1308 Asp Gly Thr Met Ser Val Glu Asp Phe Phe Tyr Gly Leu Phe Lys Asn 240 245 250 255 gga aaa tct ctt aca cca tca gca tct act cca tat aga caa cta aaa 1356 Gly Lys Ser Leu Thr Pro Ser Ala Ser Thr Pro Tyr Arg Gln Leu Lys 260 265 270 agg cac ctt tcc atg cag tct ttc gat gag agt gga cga cgt acc aca 1404 Arg His Leu Ser Met Gln Ser Phe Asp Glu Ser Gly Arg Arg Thr Thr 275 280 285 acc tca tca gca atg aca agt acc att ggc ttt cgg gtc ttc tcc tgc 1452 Thr Ser Ser Ala Met Thr Ser Thr Ile Gly Phe Arg Val Phe Ser Cys 290 295 300 ctg gat gat ggg atg ggc cat gca tct gtg gag aga ata ctg gac acc 1500 Leu Asp Asp Gly Met Gly His Ala Ser Val Glu Arg Ile Leu Asp Thr 305 310 315 tgg cag gaa gag ggc att gag aac agc cag gag atc ctg aag gcc ttg 1548 Trp Gln Glu Glu Gly Ile Glu Asn Ser Gln Glu Ile Leu Lys Ala Leu 320 325 330 335 gat ttc agc ctc gat gga aac atc aat ttg aca gaa tta aca ctg gcc 1596 Asp Phe Ser Leu Asp Gly Asn Ile Asn Leu Thr Glu Leu Thr Leu Ala 340 345 350 ctt gaa aat gaa ctt ttg gtt acc aag aac agc att cac cag gcg gct 1644 Leu Glu Asn Glu Leu Leu Val Thr Lys Asn Ser Ile His Gln Ala Ala 355 360 365 ctg gcc agc ttt aag gct gaa atc cgg cat ttg ttg gaa cga gtt gat 1692 Leu Ala Ser Phe Lys Ala Glu Ile Arg His Leu Leu Glu Arg Val Asp 370 375 380 cag gtg gtc aga gaa aaa gag aag cta cgg tca gat ctg gac aag gcc 1740 Gln Val Val Arg Glu Lys Glu Lys Leu Arg Ser Asp Leu Asp Lys Ala 385 390 395 gag aag ctc aag tct tta atg gcc tcg gag gtg gat gat cac cat gcg 1788 Glu Lys Leu Lys Ser Leu Met Ala Ser Glu Val Asp Asp His His Ala 400 405 410 415 gcc ata gag cgg cgg aat gag tac aac ctc agg aaa ctg gat gga gag 1836 Ala Ile Glu Arg Arg Asn Glu Tyr Asn Leu Arg Lys Leu Asp Gly Glu 420 425 430 tac aag gag cga ata gca gcc tta aaa aat gaa ctc cga aaa gag aga 1884 Tyr Lys Glu Arg Ile Ala Ala Leu Lys Asn Glu Leu Arg Lys Glu Arg 435 440 445 gag cag atc ctg cag cag gca ggc aag cag cgt tta gaa ctt gaa cag 1932 Glu Gln Ile Leu Gln Gln Ala Gly Lys Gln Arg Leu Glu Leu Glu Gln 450 455 460 gaa att gaa aag gca aaa aca gaa gag aac tat atc cgg gac cgc ctt 1980 Glu Ile Glu Lys Ala Lys Thr Glu Glu Asn Tyr Ile Arg Asp Arg Leu 465 470 475 gcc ctc tct tta aag gaa aac agt cgt ctg gaa aat gag ctt cta gaa 2028 Ala Leu Ser Leu Lys Glu Asn Ser Arg Leu Glu Asn Glu Leu Leu Glu 480 485 490 495 aat gca gag aag ttg gca gaa tat gag aat ctg aca aac aaa ctt cag 2076 Asn Ala Glu Lys Leu Ala Glu Tyr Glu Asn Leu Thr Asn Lys Leu Gln 500 505 510 aga aat ttg gaa aat gtg tta gca gaa aag ttt ggt gac ctc gat cct 2124 Arg Asn Leu Glu Asn Val Leu Ala Glu Lys Phe Gly Asp Leu Asp Pro 515 520 525 agc agt gct gag ttc ttc ctg caa gaa gag aga ctg aca cag atg aga 2172 Ser Ser Ala Glu Phe Phe Leu Gln Glu Glu Arg Leu Thr Gln Met Arg 530 535 540 aat gaa tat gag cgg cag tgc agg gta cta caa gac caa gta gat gaa 2220 Asn Glu Tyr Glu Arg Gln Cys Arg Val Leu Gln Asp Gln Val Asp Glu 545 550 555 ctc cag tct gag ctg gaa gaa tat cgt gca caa ggc aga gtg ctc agg 2268 Leu Gln Ser Glu Leu Glu Glu Tyr Arg Ala Gln Gly Arg Val Leu Arg 560 565 570 575 ctt ccg ttg aag aac tca ccg tca gaa gaa gtt gag gct aac agc ggt 2316 Leu Pro Leu Lys Asn Ser Pro Ser Glu Glu Val Glu Ala Asn Ser Gly 580 585 590 ggc att gag ccc gaa cac ggg ctc ggt tct gaa gaa tgc aat cca ttg 2364 Gly Ile Glu Pro Glu His Gly Leu Gly Ser Glu Glu Cys Asn Pro Leu 595 600 605 aat atg agc att gag gca gag ctg gtc att gaa cag atg aaa gaa caa 2412 Asn Met Ser Ile Glu Ala Glu Leu Val Ile Glu Gln Met Lys Glu Gln 610 615 620 cat cac agg gac ata tgt tgc ctc aga ctg gag ctc gaa gat aaa gtg 2460 His His Arg Asp Ile Cys Cys Leu Arg Leu Glu Leu Glu Asp Lys Val 625 630 635 cgc cat tat gaa aag cag ctg gac gaa acc gtg gtc agc tgc aag aag 2508 Arg His Tyr Glu Lys Gln Leu Asp Glu Thr Val Val Ser Cys Lys Lys 640 645 650 655 gca cag gag aac atg aag caa agg cat gag aac gaa acg cgc acc tta 2556 Ala Gln Glu Asn Met Lys Gln Arg His Glu Asn Glu Thr Arg Thr Leu 660 665 670 gaa aaa caa ata agt gac ctt aaa aat gaa att gct gaa ctt cag ggg 2604 Glu Lys Gln Ile Ser Asp Leu Lys Asn Glu Ile Ala Glu Leu Gln Gly 675 680 685 caa gca gca gtg ctc aag gag gca cat cat gag gcc act tgc agg cat 2652 Gln Ala Ala Val Leu Lys Glu Ala His His Glu Ala Thr Cys Arg His 690 695 700 gag gag gag aaa aaa caa ctg caa gtg aag ctt gag gag gaa aag act 2700 Glu Glu Glu Lys Lys Gln Leu Gln Val Lys Leu Glu Glu Glu Lys Thr 705 710 715 cac ctg cag gag aag ctg agg ctg caa cat gag atg gag ctc aag gct 2748 His Leu Gln Glu Lys Leu Arg Leu Gln His Glu Met Glu Leu Lys Ala 720 725 730 735 aga ctg aca cag gct caa gca agc ttt gag cgg gag agg gaa ggc ctt 2796 Arg Leu Thr Gln Ala Gln Ala Ser Phe Glu Arg Glu Arg Glu Gly Leu 740 745 750 cag agt agc gcc tgg aca gaa gag aag gtg aga ggc ttg act cag gaa 2844 Gln Ser Ser Ala Trp Thr Glu Glu Lys Val Arg Gly Leu Thr Gln Glu 755 760 765 cta gag cag ttt cac cag gag cag ctg aca agc ctg gtg gag aaa cac 2892 Leu Glu Gln Phe His Gln Glu Gln Leu Thr Ser Leu Val Glu Lys His 770 775 780 act ctt gag aaa gag gag tta aga aaa gag ctc ttg gaa aag cac caa 2940 Thr Leu Glu Lys Glu Glu Leu Arg Lys Glu Leu Leu Glu Lys His Gln 785 790 795 agg gag ctt cag gag gga agg gaa aaa atg gaa aca gag tgt aat aga 2988 Arg Glu Leu Gln Glu Gly Arg Glu Lys Met Glu Thr Glu Cys Asn Arg 800 805 810 815 aga acc tct caa ata gaa gcc cag ttt cag tct gat tgt cag aaa gtc 3036 Arg Thr Ser Gln Ile Glu Ala Gln Phe Gln Ser Asp Cys Gln Lys Val 820 825 830 act gag agg tgt gaa agc gct ctg caa agc ctg gag ggg cgc tac cgc 3084 Thr Glu Arg Cys Glu Ser Ala Leu Gln Ser Leu Glu Gly Arg Tyr Arg 835 840 845 caa gag ctg aag gac ctc cag gaa cag cag cgt gag gag aaa tcc cag 3132 Gln Glu Leu Lys Asp Leu Gln Glu Gln Gln Arg Glu Glu Lys Ser Gln 850 855 860 tgg gaa ttt gag aag gac gag ctc acc cag gag tgt gcg gaa gcc cag 3180 Trp Glu Phe Glu Lys Asp Glu Leu Thr Gln Glu Cys Ala Glu Ala Gln 865 870 875 gag ctg ctg aaa gag act ctt aag aga gag aaa aca act tct ctg gtc 3228 Glu Leu Leu Lys Glu Thr Leu Lys Arg Glu Lys Thr Thr Ser Leu Val 880 885 890 895 ctg acc cag gag aga gag atg ctg gag aaa aca tac aaa gaa cat ttg 3276 Leu Thr Gln Glu Arg Glu Met Leu Glu Lys Thr Tyr Lys Glu His Leu 900 905 910 aac agc atg gtc gtc gag aga cag cag cta ctc caa gac ctg gaa gac 3324 Asn Ser Met Val Val Glu Arg Gln Gln Leu Leu Gln Asp Leu Glu Asp 915 920 925 cta aga aat gta tct gaa acc cag caa agc ctg ctg tct gac cag ata 3372 Leu Arg Asn Val Ser Glu Thr Gln Gln Ser Leu Leu Ser Asp Gln Ile 930 935 940 ctt gag ctg aag agc agt cac aaa agg gaa ctg agg gag cgt gag gag 3420 Leu Glu Leu Lys Ser Ser His Lys Arg Glu Leu Arg Glu Arg Glu Glu 945 950 955 gtc ctg tgc cag gca ggg gct tcg gag cag ctg gcc agc cag cgg ctg 3468 Val Leu Cys Gln Ala Gly Ala Ser Glu Gln Leu Ala Ser Gln Arg Leu 960 965 970 975 gaa aga cta gaa atg gaa cat gac cag gaa agg cag gaa atg atg tcc 3516 Glu Arg Leu Glu Met Glu His Asp Gln Glu Arg Gln Glu Met Met Ser 980 985 990 aag ctt cta gcc atg gag aac att cac aaa gcg acc tgt gag aca gca 3564 Lys Leu Leu Ala Met Glu Asn Ile His Lys Ala Thr Cys Glu Thr Ala 995 1000 1005 gat cga gaa aga gcc gag atg agc aca gaa atc tcc aga ctt cag 3609 Asp Arg Glu Arg Ala Glu Met Ser Thr Glu Ile Ser Arg Leu Gln 1010 1015 1020 agt aaa ata aag gaa atg cag cag gca aca tct cct ctc tca atg 3654 Ser Lys Ile Lys Glu Met Gln Gln Ala Thr Ser Pro Leu Ser Met 1025 1030 1035 ctt cag agt ggt tgc cag gtg ata gga gag gag gag gtg gaa gga 3699 Leu Gln Ser Gly Cys Gln Val Ile Gly Glu Glu Glu Val Glu Gly 1040 1045 1050 gat gga gcc ctg tcc ctg ctt cag caa ggg gag cag ctg ttg gaa 3744 Asp Gly Ala Leu Ser Leu Leu Gln Gln Gly Glu Gln Leu Leu Glu 1055 1060 1065 gaa aat ggg gac gtc ctc tta agc ctg cag aga gct cat gaa cag 3789 Glu Asn Gly Asp Val Leu Leu Ser Leu Gln Arg Ala His Glu Gln 1070 1075 1080 gca gtg aag gaa aat gtg aaa atg gct act gaa att tct aga ttg 3834 Ala Val Lys Glu Asn Val Lys Met Ala Thr Glu Ile Ser Arg Leu 1085 1090 1095 caa cag agg cta caa aag tta gag cca ggg tta gta atg tct tct 3879 Gln Gln Arg Leu Gln Lys Leu Glu Pro Gly Leu Val Met Ser Ser 1100 1105 1110 tgt ttg gat gag cca gct act gag ttt ttt gga aat act gcg gaa 3924 Cys Leu Asp Glu Pro Ala Thr Glu Phe Phe Gly Asn Thr Ala Glu 1115 1120 1125 caa aca gag cag ttt tta cag caa aac cga acg aag caa gta gaa 3969 Gln Thr Glu Gln Phe Leu Gln Gln Asn Arg Thr Lys Gln Val Glu 1130 1135 1140 ggt gtg acc agg cgg cat gtc cta agt gac ctg gaa gat gat gag 4014 Gly Val Thr Arg Arg His Val Leu Ser Asp Leu Glu Asp Asp Glu 1145 1150 1155 gtc cgg gac ctg gga agt aca ggg acg agc tct gtt cag aga cag 4059 Val Arg Asp Leu Gly Ser Thr Gly Thr Ser Ser Val Gln Arg Gln 1160 1165 1170 gaa gtc aaa ata gag gag tct gaa gct tca gta gag ggt ttt tct 4104 Glu Val Lys Ile Glu Glu Ser Glu Ala Ser Val Glu Gly Phe Ser 1175 1180 1185 gag ctt gaa aac agt gaa gag acc agg act gaa tcc tgg gag ctg 4149 Glu Leu Glu Asn Ser Glu Glu Thr Arg Thr Glu Ser Trp Glu Leu 1190 1195 1200 aag aat cag att agt cag ctt cag gaa cag cta atg atg tta tgt 4194 Lys Asn Gln Ile Ser Gln Leu Gln Glu Gln Leu Met Met Leu Cys 1205 1210 1215 gcg gac tgt gat cga gct tct gaa aag aaa cag gac cta ctt ttt 4239 Ala Asp Cys Asp Arg Ala Ser Glu Lys Lys Gln Asp Leu Leu Phe 1220 1225 1230 gat gtt tct gtg cta aaa aag aaa ctg aag atg ctt gag aga atc 4284 Asp Val Ser Val Leu Lys Lys Lys Leu Lys Met Leu Glu Arg Ile 1235 1240 1245 cct gag gct tct ccc aaa tat aag ctg ttg tat gaa gat gtg agc 4329 Pro Glu Ala Ser Pro Lys Tyr Lys Leu Leu Tyr Glu Asp Val Ser 1250 1255 1260 cga gaa aat gac tgc ctt cag gaa gag ctg aga atg atg gag aca 4374 Arg Glu Asn Asp Cys Leu Gln Glu Glu Leu Arg Met Met Glu Thr 1265 1270 1275 cgc tac gat gag gca cta gaa aat aac aaa gaa ctc act gca gag 4419 Arg Tyr Asp Glu Ala Leu Glu Asn Asn Lys Glu Leu Thr Ala Glu 1280 1285 1290 gtt ttc agg ttg cag gat gag ctg aag aaa atg gag gaa gtc act 4464 Val Phe Arg Leu Gln Asp Glu Leu Lys Lys Met Glu Glu Val Thr 1295 1300 1305 gaa aca ttc ctc agc ctg gaa aag agt tac gat gag gtc aaa ata 4509 Glu Thr Phe Leu Ser Leu Glu Lys Ser Tyr Asp Glu Val Lys Ile 1310 1315 1320 gaa aat gag ggg ctg aat gtt ctg gtt ttg aga ctt caa ggc aag 4554 Glu Asn Glu Gly Leu Asn Val Leu Val Leu Arg Leu Gln Gly Lys 1325 1330 1335 att gag aag ctt cag gaa agc gtg gtc cag cgg tgt gac tgc tgc 4599 Ile Glu Lys Leu Gln Glu Ser Val Val Gln Arg Cys Asp Cys Cys 1340 1345 1350 tta tgg gaa gcc agt tta gag aac ctg gaa atc gaa cct gat gga 4644 Leu Trp Glu Ala Ser Leu Glu Asn Leu Glu Ile Glu Pro Asp Gly 1355 1360 1365 aat ata ctc cag ctc aat cag aca ctg gaa gag tgt gtg ccc agg 4689 Asn Ile Leu Gln Leu Asn Gln Thr Leu Glu Glu Cys Val Pro Arg 1370 1375 1380 gtt agg agt gta cat cat gtc ata gag gaa tgt aag caa gaa aac 4734 Val Arg Ser Val His His Val Ile Glu Glu Cys Lys Gln Glu Asn 1385 1390 1395 cag tac ctt gag ggg aac aca cag ctc ttg gaa aaa gta aaa gca 4779 Gln Tyr Leu Glu Gly Asn Thr Gln Leu Leu Glu Lys Val Lys Ala 1400 1405 1410 cat gaa att gcc tgg tta cat gga aca att cag aca cat caa gaa 4824 His Glu Ile Ala Trp Leu His Gly Thr Ile Gln Thr His Gln Glu 1415 1420 1425 agg cca aga gta cag aat caa gtt ata ctg gag gaa aac act act 4869 Arg Pro Arg Val Gln Asn Gln Val Ile Leu Glu Glu Asn Thr Thr 1430 1435 1440 ctc cta ggc ttt caa gac aaa cat ttt cag cat cag gcc acc ata 4914 Leu Leu Gly Phe Gln Asp Lys His Phe Gln His Gln Ala Thr Ile 1445 1450 1455 gca gag tta gaa ctg gag aaa aca aag tta cag gag ctg act agg 4959 Ala Glu Leu Glu Leu Glu Lys Thr Lys Leu Gln Glu Leu Thr Arg 1460 1465 1470 aag ttg aag gag aga gtc act att tta gtt aag caa aaa gat gta 5004 Lys Leu Lys Glu Arg Val Thr Ile Leu Val Lys Gln Lys Asp Val 1475 1480 1485 ctt tct cac gga gaa aag gag gaa gag ctg aag gca atg atg cat 5049 Leu Ser His Gly Glu Lys Glu Glu Glu Leu Lys Ala Met Met His 1490 1495 1500 gac ttg cag atc acg tgc agt gag atg cag caa aaa gtt gaa ctt 5094 Asp Leu Gln Ile Thr Cys Ser Glu Met Gln Gln Lys Val Glu Leu 1505 1510 1515 ctg aga tat gaa tct gaa aag ctt caa cag gaa aat tct att ttg 5139 Leu Arg Tyr Glu Ser Glu Lys Leu Gln Gln Glu Asn Ser Ile Leu 1520 1525 1530 aga aat gaa att act act tta aat gaa gaa gat agc att tct aac 5184 Arg Asn Glu Ile Thr Thr Leu Asn Glu Glu Asp Ser Ile Ser Asn 1535 1540 1545 ctg aaa tta ggg aca tta aat gga tct cag gaa gaa atg tgg caa 5229 Leu Lys Leu Gly Thr Leu Asn Gly Ser Gln Glu Glu Met Trp Gln 1550 1555 1560 aaa acg gaa act gta aaa caa gaa aat gct gca gtt cag aag atg 5274 Lys Thr Glu Thr Val Lys Gln Glu Asn Ala Ala Val Gln Lys Met 1565 1570 1575 gtt gaa aat tta aag aaa cag att tca gaa tta aaa atc aaa aac 5319 Val Glu Asn Leu Lys Lys Gln Ile Ser Glu Leu Lys Ile Lys Asn 1580 1585 1590 caa caa ttg gat ttg gaa aat aca gaa ctt agc caa aag aac tct 5364 Gln Gln Leu Asp Leu Glu Asn Thr Glu Leu Ser Gln Lys Asn Ser 1595 1600 1605 caa aac cag gaa aaa ctg caa gaa ctt aat caa cgt cta aca gaa 5409 Gln Asn Gln Glu Lys Leu Gln Glu Leu Asn Gln Arg Leu Thr Glu 1610 1615 1620 atg cta tgc cag aag gaa aaa gag cca gga aac agt gca ttg gag 5454 Met Leu Cys Gln Lys Glu Lys Glu Pro Gly Asn Ser Ala Leu Glu 1625 1630 1635 gaa cgg gaa caa gag aag ttt aat ctg aaa gaa gaa ctg gaa cgt 5499 Glu Arg Glu Gln Glu Lys Phe Asn Leu Lys Glu Glu Leu Glu Arg 1640 1645 1650 tgt aaa gtg cag tcc tcc act tta gtg tct tct ctg gag gcg gag 5544 Cys Lys Val Gln Ser Ser Thr Leu Val Ser Ser Leu Glu Ala Glu 1655 1660 1665 ctc tct gaa gtt aaa ata cag acc cat att gtg caa cag gaa aac 5589 Leu Ser Glu Val Lys Ile Gln Thr His Ile Val Gln Gln Glu Asn 1670 1675 1680 cac ctt ctc aaa gat gaa ctg gag aaa atg aaa cag ctg cac aga 5634 His Leu Leu Lys Asp Glu Leu Glu Lys Met Lys Gln Leu His Arg 1685 1690 1695 tgt ccc gat ctc tct gac ttc cag caa aaa atc tct agt gtt cta 5679 Cys Pro Asp Leu Ser Asp Phe Gln Gln Lys Ile Ser Ser Val Leu 1700 1705 1710 agc tac aac gaa aaa ctg ctg aaa gaa aag gaa gct ctg agt gag 5724 Ser Tyr Asn Glu Lys Leu Leu Lys Glu Lys Glu Ala Leu Ser Glu 1715 1720 1725 gaa tta aat agc tgt gtc gat aag ttg gca aaa tca agt ctt tta 5769 Glu Leu Asn Ser Cys Val Asp Lys Leu Ala Lys Ser Ser Leu Leu 1730 1735 1740 gag cat aga att gcg acg atg aag cag gaa cag aaa tcc tgg gaa 5814 Glu His Arg Ile Ala Thr Met Lys Gln Glu Gln Lys Ser Trp Glu 1745 1750 1755 cat cag agt gcg agc tta aag tca cag ctg gtg gct tct cag gaa 5859 His Gln Ser Ala Ser Leu Lys Ser Gln Leu Val Ala Ser Gln Glu 1760 1765 1770 aag gtt cag aat tta gaa gac acc gtg cag aat gta aac ctg caa 5904 Lys Val Gln Asn Leu Glu Asp Thr Val Gln Asn Val Asn Leu Gln 1775 1780 1785 atg tcc cgg atg aaa tct gac cta cga gtg act cag cag gaa aag 5949 Met Ser Arg Met Lys Ser Asp Leu Arg Val Thr Gln Gln Glu Lys 1790 1795 1800 gag gct tta aaa caa gaa gtg atg tct tta cat aag caa ctt cag 5994 Glu Ala Leu Lys Gln Glu Val Met Ser Leu His Lys Gln Leu Gln 1805 1810 1815 aat gct ggt ggc aag agc tgg gcc cca gag ata gct act cat cca 6039 Asn Ala Gly Gly Lys Ser Trp Ala Pro Glu Ile Ala Thr His Pro 1820 1825 1830 tca ggg ctc cat aac cag cag aaa agg ctg tcc tgg gac aag ttg 6084 Ser Gly Leu His Asn Gln Gln Lys Arg Leu Ser Trp Asp Lys Leu 1835 1840 1845 gat cat ctg atg aat gag gaa cag cag ctg ctt tgg caa gag aat 6129 Asp His Leu Met Asn Glu Glu Gln Gln Leu Leu Trp Gln Glu Asn 1850 1855 1860 gag agg ctc cag acc atg gta cag aac acc aaa gcc gaa ctc acg 6174 Glu Arg Leu Gln Thr Met Val Gln Asn Thr Lys Ala Glu Leu Thr 1865 1870 1875 cac tcc cgg gag aag gtc cgt caa ttg gaa tcc aat ctt ctt ccc 6219 His Ser Arg Glu Lys Val Arg Gln Leu Glu Ser Asn Leu Leu Pro 1880 1885 1890 aag cac caa aaa cat cta aac cca tca ggt acc atg aat ccc aca 6264 Lys His Gln Lys His Leu Asn Pro Ser Gly Thr Met Asn Pro Thr 1895 1900 1905 gag caa gaa aaa ttg agc tta aag aga gag tgt gat cag ttt cag 6309 Glu Gln Glu Lys Leu Ser Leu Lys Arg Glu Cys Asp Gln Phe Gln 1910 1915 1920 aaa gaa caa tct cct gct aac agg aag gtc agt cag atg aat tcc 6354 Lys Glu Gln Ser Pro Ala Asn Arg Lys Val Ser Gln Met Asn Ser 1925 1930 1935 ctt gaa caa gaa tta gaa aca att cat ttg gaa aat gaa ggc ctg 6399 Leu Glu Gln Glu Leu Glu Thr Ile His Leu Glu Asn Glu Gly Leu 1940 1945 1950 aaa aag aaa caa gta aaa ctg gat gag cag ctc atg gag atg cag 6444 Lys Lys Lys Gln Val Lys Leu Asp Glu Gln Leu Met Glu Met Gln 1955 1960 1965 cac ctg agg tcc act gcg acg cct agc ccg tcc cct cat gct tgg 6489 His Leu Arg Ser Thr Ala Thr Pro Ser Pro Ser Pro His Ala Trp 1970 1975 1980 gat ttg cag ctg ctc cag cag caa gcc tgt ccg atg gtg ccc agg 6534 Asp Leu Gln Leu Leu Gln Gln Gln Ala Cys Pro Met Val Pro Arg 1985 1990 1995 gag cag ttt ctg cag ctt caa cgc cag ctg ctg cag gca gaa agg 6579 Glu Gln Phe Leu Gln Leu Gln Arg Gln Leu Leu Gln Ala Glu Arg 2000 2005 2010 ata aac cag cac ctg cag gag gaa ctt gaa aac agg acc tcc gaa 6624 Ile Asn Gln His Leu Gln Glu Glu Leu Glu Asn Arg Thr Ser Glu 2015 2020 2025 acc aac aca cca cag gga aac cag gaa caa ctg gta act gtc atg 6669 Thr Asn Thr Pro Gln Gly Asn Gln Glu Gln Leu Val Thr Val Met 2030 2035 2040 gag gaa cga atg ata gaa gtt gaa cag aaa ctg aaa cta gtg aaa 6714 Glu Glu Arg Met Ile Glu Val Glu Gln Lys Leu Lys Leu Val Lys 2045 2050 2055 agg ctt ctt caa gag aaa gtg aat cag ctc aaa gaa caa ctc tgc 6759 Arg Leu Leu Gln Glu Lys Val Asn Gln Leu Lys Glu Gln Leu Cys 2060 2065 2070 aag aac act aag gca gac gca atg gtg aag gac ttg tat gtt gaa 6804 Lys Asn Thr Lys Ala Asp Ala Met Val Lys Asp Leu Tyr Val Glu 2075 2080 2085 aat gcc cag ttg ttg aaa gct ctg gaa gtg act gaa cag cga cag 6849 Asn Ala Gln Leu Leu Lys Ala Leu Glu Val Thr Glu Gln Arg Gln 2090 2095 2100 aaa aca gca gag aag aaa aat tac ctc ctg gag gag aag att gcc 6894 Lys Thr Ala Glu Lys Lys Asn Tyr Leu Leu Glu Glu Lys Ile Ala 2105 2110 2115 agc ctc agt aat ata gtt agg aat ctg aca cca gcg cca ttg act 6939 Ser Leu Ser Asn Ile Val Arg Asn Leu Thr Pro Ala Pro Leu Thr 2120 2125 2130 tct aca cct cct ttg agg tca tagccaaacc aaagggtaca ctcatatttg 6990 Ser Thr Pro Pro Leu Arg Ser 2135 tgcactttac tgaaatagat gaacatttca gtaggttctc aacttaaaat taagcctaac 7050 ctaaaactgc cagcaacaca actggagttt ccatttatca taattagttt ttctaaatag 7110 acccttatgg gagtttgaaa ataaatactc acatatttca ctacttaaat tattcccaag 7170 atttgaattt attttaaaat tttaatagcc accaagaatg tggacatatg aaaattcaag 7230 aacctaaaaa ataccagttt tgaatgagtt tttgtggttt tggtttttta attattacaa 7290 atctatgtgt aaaatctaga tatttgaagt ttgagatctg atgagaatgg ttgttataaa 7350 ctttatttta aaaccaaatt taggtgttct tacatattta aatactggaa agtcattata 7410 atagttttgg ttctttgaat tggtagacaa ttagtagagt ataattggtt aggaggcagg 7470 gcttattaag tggttattaa ccgctgacat cagacaaacc caaatctgta gaattctaac 7530 ctcctaacac ctgtgacagt attaccactc ttcttgtatt atagatttag aactgattta 7590 ctcaattgca ctcttaacta atgttaaaag cttacttgct ttaaacagcc ttttcttctt 7650 tctcttaaaa gtttcatttg gggagctggt cttctaagaa acggataaag ccacataatt 7710 aaagcagttg aactagaggg aaagcactga acaaaccact ttggagtaaa tagctactct 7770 tagaaaagag ggataagcag accatgtagg ttttctgtct ctcaaatctt agagttcata 7830 aatttacttg aggttgcctc aagaactcag ggaacaatac tgtaaactgt cttcctgaac 7890 tactgtaggg cctctctaag aatttgaaat gtataaacca tgtgacctca tttatttgtc 7950 ttatatattt acagccatac tagaattttt atttctacgt ttttagtaaa tttaatattc 8010 tgggggaaaa aaggccttga ttttagggtt aaaaacctga cttatagaag agtttattta 8070 atataggtca aaattttctg tgtttcttat tccttctata cctcaaatct gattctaaga 8130 atttcttact gtgataatca ttggcatgcc acctgaggtc aaggagtgcc aaataggact 8190 ttccactcat gctcaagatc aaaactttat agaacagtca acattttaga ttcggtaacc 8250 ttttttttct tccaattata atctctgctt ctagccactt ccgccagcag ttggtggaag 8310 acttactagg tgcagggcac tttccaagtt catcacaaca acctgcttgt tttcatgaga 8370 caataatccg aaaagttcgc tttgatatat tcctggaggg ccaagcccat ctatttacaa 8430 aaggtgaaca gcaaaatcaa gcactgcttt atgggcagga acacaagaga aagcaaactg 8490 cccaagaagt catcatgtca gaaactcaat ctcaacaaaa taatttccat cagggaactt 8550 cagggtttct tgggggctta tgagtctcac cggtcaaccc aggaggcctc actacaagag 8610 ccttgacaag gcactgtttt ttgtgggact gggagttcac actgatgaag caaacctttg 8670 aatttttgca cagctcttgt cagaaagccc tgagttcccc ctggataaag agttaatttt 8730 aatccttccc tataattata cttcaaaata tttgacatct gctattatgc cttctttaga 8790 tctttcttct gcggtgcaga catttctagt aagtgtttga ctacttgtat ggcattagct 8850 ttcacagaaa attgtttcac ttaaaactgt ggattggcct aggctaagga caaaaataaa 8910 ctaagtacct gtagtgtatt tatgtgatat gtgtcaagtt actcaaagtt attgctgttg 8970 gaactgaaca ataatatttc ccagatagct ggccttagca tgtgatcacg gttgttgtat 9030 ttttaatttt tgtcttttac agtatgagag gtgtaggtta atttgtttat ttcctataaa 9090 tttgtattta tgtgtatata aaatgtacaa tgaatgtaaa tatgactttc tggaaagttt 9150 agactacatt tagaatctct attcaaaatc aaaatgctgc tcaaatgaat ttaaccaaca 9210 tctaggtgct taatttctca ttttatccca cttatgagat tgggaaaaag atcaatatga 9270 gaaataccat acagatacct taaatgtatg catttgtgca acaatttttg agaaggtgag 9330 tggcaattta taatttagtt ggcaatttat aatagaactt atagctttta aaagactttt 9390 taaagacatt aaatgtaaac ttaaaaatgt ttagatcttg tttcaaactt tacaatagca 9450 ttcttcaaaa tattaagtta tatattttat aggcatttag ttgcttatta aaagcactga 9510 ttttcaaact ttttgattta agaacaatta tttaagatcg tctcagaaga tgggatcttc 9570 gtttcaagaa aagggaatca agtttgcctt tgagataata cgttacacta agaaaaggaa 9630 aatgtggata gtaaaaccca cctctctcat cctattgtac tctcttctgc tttttagaag 9690 cctgcactta agcttagatt tgtgaaggga gagtagaagg ggagaagtag aaccacagtg 9750 ttttatttat ttttctaaaa ctcttactaa atccagattt tttaaactgt tttaaatgtg 9810 aattcttccc agaaatttca atgcattgca tatttagcct tcggcatatt tttcatgaat 9870 agatcatgaa gtcataggct tccaaggcat aggaagagat cttgcaggtc tagtatttta 9930 ataatgcact attacccagg gcagatatta tgagaaactg tttcttctct aagggtttat 9990 ggcagacttt gcttttttaa catgtgagaa atgaattttt tattttgtga tttatgtgat 10050 ttcttttgct gagtgaagga aaggagaaat tgttgctatt gtcagcatct taaaggtatt 10110 tccagtcaag gcaaggctaa gtgctttgtg atagtattaa gcaagtcatg ttttgaatgg 10170 attacctgta gtgactcatt ggaatgatat aattatacaa gtaatgccaa aaaccaagtc 10230 aaagcctaat taaccaaagc actcatttaa aaatcatcat gtttggacct atctggacct 10290 ctcagcactg taaaatagtt ttggttttgt ggcatatgaa tagctgttta acaaatcaaa 10350 gttagctttt tgcttctcag cttttttggg caatacaagt taagttctta atggggagac 10410 attatcatgg catgacttaa gggaacattg gtttgtgaag gaaaaacaga ttatctaaag 10470 ccatctctat gtttctgttc agataaagat taatgagttc tgtgtttata tcagctttgt 10530 atatttcatc ttagccattc tatcctagaa agattttaat gtgagcttaa gatgtaaata 10590 aataattttg caaacatgaa aaaaaaaaaa aaaaa 10625 6 2139 PRT Homo sapiens 6 Met Ala Glu Val Thr Val Pro Arg Val Tyr Val Val Phe Gly Ile His 1 5 10 15 Cys Ile Met Ala Lys Ala Ser Ser Asp Val Gln Val Ser Gly Phe His 20 25 30 Arg Lys Ile Gln His Val Lys Asn Glu Leu Cys His Met Leu Ser Leu 35 40 45 Glu Glu Val Ala Pro Val Leu Gln Gln Thr Leu Leu Gln Asp Asn Leu 50 55 60 Leu Gly Arg Val His Phe Asp Gln Phe Lys Glu Ala Leu Ile Leu Ile 65 70 75 80 Leu Ser Arg Thr Leu Ser Asn Glu Glu His Phe Gln Glu Pro Asp Cys 85 90 95 Ser Leu Glu Ala Gln Pro Lys Tyr Val Arg Gly Gly Lys Arg Tyr Gly 100 105 110 Arg Arg Ser Leu Pro Glu Phe Gln Glu Ser Val Glu Glu Phe Pro Glu 115 120 125 Val Thr Val Ile Glu Pro Leu Asp Glu Glu Ala Arg Pro Ser His Ile 130 135 140 Pro Ala Gly Asp Cys Ser Glu His Trp Lys Thr Gln Arg Ser Glu Glu 145 150 155 160 Tyr Glu Ala Glu Gly Gln Leu Arg Phe Trp Asn Pro Asp Asp Leu Asn 165 170 175 Ala Ser Gln Ser Gly Ser Ser Pro Pro Gln Asp Trp Ile Glu Glu Lys 180 185 190 Leu Gln Glu Val Cys Glu Asp Leu Gly Ile Thr Arg Asp Gly His Leu 195 200 205 Asn Arg Lys Lys Leu Val Ser Ile Cys Glu Gln Tyr Gly Leu Gln Asn 210 215 220 Val Asp Gly Glu Met Leu Glu Glu Val Phe His Asn Leu Asp Pro Asp 225 230 235 240 Gly Thr Met Ser Val Glu Asp Phe Phe Tyr Gly Leu Phe Lys Asn Gly 245 250 255 Lys Ser Leu Thr Pro Ser Ala Ser Thr Pro Tyr Arg Gln Leu Lys Arg 260 265 270 His Leu Ser Met Gln Ser Phe Asp Glu Ser Gly Arg Arg Thr Thr Thr 275 280 285 Ser Ser Ala Met Thr Ser Thr Ile Gly Phe Arg Val Phe Ser Cys Leu 290 295 300 Asp Asp Gly Met Gly His Ala Ser Val Glu Arg Ile Leu Asp Thr Trp 305 310 315 320 Gln Glu Glu Gly Ile Glu Asn Ser Gln Glu Ile Leu Lys Ala Leu Asp 325 330 335 Phe Ser Leu Asp Gly Asn Ile Asn Leu Thr Glu Leu Thr Leu Ala Leu 340 345 350 Glu Asn Glu Leu Leu Val Thr Lys Asn Ser Ile His Gln Ala Ala Leu 355 360 365 Ala Ser Phe Lys Ala Glu Ile Arg His Leu Leu Glu Arg Val Asp Gln 370 375 380 Val Val Arg Glu Lys Glu Lys Leu Arg Ser Asp Leu Asp Lys Ala Glu 385 390 395 400 Lys Leu Lys Ser Leu Met Ala Ser Glu Val Asp Asp His His Ala Ala 405 410 415 Ile Glu Arg Arg Asn Glu Tyr Asn Leu Arg Lys Leu Asp Gly Glu Tyr 420 425 430 Lys Glu Arg Ile Ala Ala Leu Lys Asn Glu Leu Arg Lys Glu Arg Glu 435 440 445 Gln Ile Leu Gln Gln Ala Gly Lys Gln Arg Leu Glu Leu Glu Gln Glu 450 455 460 Ile Glu Lys Ala Lys Thr Glu Glu Asn Tyr Ile Arg Asp Arg Leu Ala 465 470 475 480 Leu Ser Leu Lys Glu Asn Ser Arg Leu Glu Asn Glu Leu Leu Glu Asn 485 490 495 Ala Glu Lys Leu Ala Glu Tyr Glu Asn Leu Thr Asn Lys Leu Gln Arg 500 505 510 Asn Leu Glu Asn Val Leu Ala Glu Lys Phe Gly Asp Leu Asp Pro Ser 515 520 525 Ser Ala Glu Phe Phe Leu Gln Glu Glu Arg Leu Thr Gln Met Arg Asn 530 535 540 Glu Tyr Glu Arg Gln Cys Arg Val Leu Gln Asp Gln Val Asp Glu Leu 545 550 555 560 Gln Ser Glu Leu Glu Glu Tyr Arg Ala Gln Gly Arg Val Leu Arg Leu 565 570 575 Pro Leu Lys Asn Ser Pro Ser Glu Glu Val Glu Ala Asn Ser Gly Gly 580 585 590 Ile Glu Pro Glu His Gly Leu Gly Ser Glu Glu Cys Asn Pro Leu Asn 595 600 605 Met Ser Ile Glu Ala Glu Leu Val Ile Glu Gln Met Lys Glu Gln His 610 615 620 His Arg Asp Ile Cys Cys Leu Arg Leu Glu Leu Glu Asp Lys Val Arg 625 630 635 640 His Tyr Glu Lys Gln Leu Asp Glu Thr Val Val Ser Cys Lys Lys Ala 645 650 655 Gln Glu Asn Met Lys Gln Arg His Glu Asn Glu Thr Arg Thr Leu Glu 660 665 670 Lys Gln Ile Ser Asp Leu Lys Asn Glu Ile Ala Glu Leu Gln Gly Gln 675 680 685 Ala Ala Val Leu Lys Glu Ala His His Glu Ala Thr Cys Arg His Glu 690 695 700 Glu Glu Lys Lys Gln Leu Gln Val Lys Leu Glu Glu Glu Lys Thr His 705 710 715 720 Leu Gln Glu Lys Leu Arg Leu Gln His Glu Met Glu Leu Lys Ala Arg 725 730 735 Leu Thr Gln Ala Gln Ala Ser Phe Glu Arg Glu Arg Glu Gly Leu Gln 740 745 750 Ser Ser Ala Trp Thr Glu Glu Lys Val Arg Gly Leu Thr Gln Glu Leu 755 760 765 Glu Gln Phe His Gln Glu Gln Leu Thr Ser Leu Val Glu Lys His Thr 770 775 780 Leu Glu Lys Glu Glu Leu Arg Lys Glu Leu Leu Glu Lys His Gln Arg 785 790 795 800 Glu Leu Gln Glu Gly Arg Glu Lys Met Glu Thr Glu Cys Asn Arg Arg 805 810 815 Thr Ser Gln Ile Glu Ala Gln Phe Gln Ser Asp Cys Gln Lys Val Thr 820 825 830 Glu Arg Cys Glu Ser Ala Leu Gln Ser Leu Glu Gly Arg Tyr Arg Gln 835 840 845 Glu Leu Lys Asp Leu Gln Glu Gln Gln Arg Glu Glu Lys Ser Gln Trp 850 855 860 Glu Phe Glu Lys Asp Glu Leu Thr Gln Glu Cys Ala Glu Ala Gln Glu 865 870 875 880 Leu Leu Lys Glu Thr Leu Lys Arg Glu Lys Thr Thr Ser Leu Val Leu 885 890 895 Thr Gln Glu Arg Glu Met Leu Glu Lys Thr Tyr Lys Glu His Leu Asn 900 905 910 Ser Met Val Val Glu Arg Gln Gln Leu Leu Gln Asp Leu Glu Asp Leu 915 920 925 Arg Asn Val Ser Glu Thr Gln Gln Ser Leu Leu Ser Asp Gln Ile Leu 930 935 940 Glu Leu Lys Ser Ser His Lys Arg Glu Leu Arg Glu Arg Glu Glu Val 945 950 955 960 Leu Cys Gln Ala Gly Ala Ser Glu Gln Leu Ala Ser Gln Arg Leu Glu 965 970 975 Arg Leu Glu Met Glu His Asp Gln Glu Arg Gln Glu Met Met Ser Lys 980 985 990 Leu Leu Ala Met Glu Asn Ile His Lys Ala Thr Cys Glu Thr Ala Asp 995 1000 1005 Arg Glu Arg Ala Glu Met Ser Thr Glu Ile Ser Arg Leu Gln Ser 1010 1015 1020 Lys Ile Lys Glu Met Gln Gln Ala Thr Ser Pro Leu Ser Met Leu 1025 1030 1035 Gln Ser Gly Cys Gln Val Ile Gly Glu Glu Glu Val Glu Gly Asp 1040 1045 1050 Gly Ala Leu Ser Leu Leu Gln Gln Gly Glu Gln Leu Leu Glu Glu 1055 1060 1065 Asn Gly Asp Val Leu Leu Ser Leu Gln Arg Ala His Glu Gln Ala 1070 1075 1080 Val Lys Glu Asn Val Lys Met Ala Thr Glu Ile Ser Arg Leu Gln 1085 1090 1095 Gln Arg Leu Gln Lys Leu Glu Pro Gly Leu Val Met Ser Ser Cys 1100 1105 1110 Leu Asp Glu Pro Ala Thr Glu Phe Phe Gly Asn Thr Ala Glu Gln 1115 1120 1125 Thr Glu Gln Phe Leu Gln Gln Asn Arg Thr Lys Gln Val Glu Gly 1130 1135 1140 Val Thr Arg Arg His Val Leu Ser Asp Leu Glu Asp Asp Glu Val 1145 1150 1155 Arg Asp Leu Gly Ser Thr Gly Thr Ser Ser Val Gln Arg Gln Glu 1160 1165 1170 Val Lys Ile Glu Glu Ser Glu Ala Ser Val Glu Gly Phe Ser Glu 1175 1180 1185 Leu Glu Asn Ser Glu Glu Thr Arg Thr Glu Ser Trp Glu Leu Lys 1190 1195 1200 Asn Gln Ile Ser Gln Leu Gln Glu Gln Leu Met Met Leu Cys Ala 1205 1210 1215 Asp Cys Asp Arg Ala Ser Glu Lys Lys Gln Asp Leu Leu Phe Asp 1220 1225 1230 Val Ser Val Leu Lys Lys Lys Leu Lys Met Leu Glu Arg Ile Pro 1235 1240 1245 Glu Ala Ser Pro Lys Tyr Lys Leu Leu Tyr Glu Asp Val Ser Arg 1250 1255 1260 Glu Asn Asp Cys Leu Gln Glu Glu Leu Arg Met Met Glu Thr Arg 1265 1270 1275 Tyr Asp Glu Ala Leu Glu Asn Asn Lys Glu Leu Thr Ala Glu Val 1280 1285 1290 Phe Arg Leu Gln Asp Glu Leu Lys Lys Met Glu Glu Val Thr Glu 1295 1300 1305 Thr Phe Leu Ser Leu Glu Lys Ser Tyr Asp Glu Val Lys Ile Glu 1310 1315 1320 Asn Glu Gly Leu Asn Val Leu Val Leu Arg Leu Gln Gly Lys Ile 1325 1330 1335 Glu Lys Leu Gln Glu Ser Val Val Gln Arg Cys Asp Cys Cys Leu 1340 1345 1350 Trp Glu Ala Ser Leu Glu Asn Leu Glu Ile Glu Pro Asp Gly Asn 1355 1360 1365 Ile Leu Gln Leu Asn Gln Thr Leu Glu Glu Cys Val Pro Arg Val 1370 1375 1380 Arg Ser Val His His Val Ile Glu Glu Cys Lys Gln Glu Asn Gln 1385 1390 1395 Tyr Leu Glu Gly Asn Thr Gln Leu Leu Glu Lys Val Lys Ala His 1400 1405 1410 Glu Ile Ala Trp Leu His Gly Thr Ile Gln Thr His Gln Glu Arg 1415 1420 1425 Pro Arg Val Gln Asn Gln Val Ile Leu Glu Glu Asn Thr Thr Leu 1430 1435 1440 Leu Gly Phe Gln Asp Lys His Phe Gln His Gln Ala Thr Ile Ala 1445 1450 1455 Glu Leu Glu Leu Glu Lys Thr Lys Leu Gln Glu Leu Thr Arg Lys 1460 1465 1470 Leu Lys Glu Arg Val Thr Ile Leu Val Lys Gln Lys Asp Val Leu 1475 1480 1485 Ser His Gly Glu Lys Glu Glu Glu Leu Lys Ala Met Met His Asp 1490 1495 1500 Leu Gln Ile Thr Cys Ser Glu Met Gln Gln Lys Val Glu Leu Leu 1505 1510 1515 Arg Tyr Glu Ser Glu Lys Leu Gln Gln Glu Asn Ser Ile Leu Arg 1520 1525 1530 Asn Glu Ile Thr Thr Leu Asn Glu Glu Asp Ser Ile Ser Asn Leu 1535 1540 1545 Lys Leu Gly Thr Leu Asn Gly Ser Gln Glu Glu Met Trp Gln Lys 1550 1555 1560 Thr Glu Thr Val Lys Gln Glu Asn Ala Ala Val Gln Lys Met Val 1565 1570 1575 Glu Asn Leu Lys Lys Gln Ile Ser Glu Leu Lys Ile Lys Asn Gln 1580 1585 1590 Gln Leu Asp Leu Glu Asn Thr Glu Leu Ser Gln Lys Asn Ser Gln 1595 1600 1605 Asn Gln Glu Lys Leu Gln Glu Leu Asn Gln Arg Leu Thr Glu Met 1610 1615 1620 Leu Cys Gln Lys Glu Lys Glu Pro Gly Asn Ser Ala Leu Glu Glu 1625 1630 1635 Arg Glu Gln Glu Lys Phe Asn Leu Lys Glu Glu Leu Glu Arg Cys 1640 1645 1650 Lys Val Gln Ser Ser Thr Leu Val Ser Ser Leu Glu Ala Glu Leu 1655 1660 1665 Ser Glu Val Lys Ile Gln Thr His Ile Val Gln Gln Glu Asn His 1670 1675 1680 Leu Leu Lys Asp Glu Leu Glu Lys Met Lys Gln Leu His Arg Cys 1685 1690 1695 Pro Asp Leu Ser Asp Phe Gln Gln Lys Ile Ser Ser Val Leu Ser 1700 1705 1710 Tyr Asn Glu Lys Leu Leu Lys Glu Lys Glu Ala Leu Ser Glu Glu 1715 1720 1725 Leu Asn Ser Cys Val Asp Lys Leu Ala Lys Ser Ser Leu Leu Glu 1730 1735 1740 His Arg Ile Ala Thr Met Lys Gln Glu Gln Lys Ser Trp Glu His 1745 1750 1755 Gln Ser Ala Ser Leu Lys Ser Gln Leu Val Ala Ser Gln Glu Lys 1760 1765 1770 Val Gln Asn Leu Glu Asp Thr Val Gln Asn Val Asn Leu Gln Met 1775 1780 1785 Ser Arg Met Lys Ser Asp Leu Arg Val Thr Gln Gln Glu Lys Glu 1790 1795 1800 Ala Leu Lys Gln Glu Val Met Ser Leu His Lys Gln Leu Gln Asn 1805 1810 1815 Ala Gly Gly Lys Ser Trp Ala Pro Glu Ile Ala Thr His Pro Ser 1820 1825 1830 Gly Leu His Asn Gln Gln Lys Arg Leu Ser Trp Asp Lys Leu Asp 1835 1840 1845 His Leu Met Asn Glu Glu Gln Gln Leu Leu Trp Gln Glu Asn Glu 1850 1855 1860 Arg Leu Gln Thr Met Val Gln Asn Thr Lys Ala Glu Leu Thr His 1865 1870 1875 Ser Arg Glu Lys Val Arg Gln Leu Glu Ser Asn Leu Leu Pro Lys 1880 1885 1890 His Gln Lys His Leu Asn Pro Ser Gly Thr Met Asn Pro Thr Glu 1895 1900 1905 Gln Glu Lys Leu Ser Leu Lys Arg Glu Cys Asp Gln Phe Gln Lys 1910 1915 1920 Glu Gln Ser Pro Ala Asn Arg Lys Val Ser Gln Met Asn Ser Leu 1925 1930 1935 Glu Gln Glu Leu Glu Thr Ile His Leu Glu Asn Glu Gly Leu Lys 1940 1945 1950 Lys Lys Gln Val Lys Leu Asp Glu Gln Leu Met Glu Met Gln His 1955 1960 1965 Leu Arg Ser Thr Ala Thr Pro Ser Pro Ser Pro His Ala Trp Asp 1970 1975 1980 Leu Gln Leu Leu Gln Gln Gln Ala Cys Pro Met Val Pro Arg Glu 1985 1990 1995 Gln Phe Leu Gln Leu Gln Arg Gln Leu Leu Gln Ala Glu Arg Ile 2000 2005 2010 Asn Gln His Leu Gln Glu Glu Leu Glu Asn Arg Thr Ser Glu Thr 2015 2020 2025 Asn Thr Pro Gln Gly Asn Gln Glu Gln Leu Val Thr Val Met Glu 2030 2035 2040 Glu Arg Met Ile Glu Val Glu Gln Lys Leu Lys Leu Val Lys Arg 2045 2050 2055 Leu Leu Gln Glu Lys Val Asn Gln Leu Lys Glu Gln Leu Cys Lys 2060 2065 2070 Asn Thr Lys Ala Asp Ala Met Val Lys Asp Leu Tyr Val Glu Asn 2075 2080 2085 Ala Gln Leu Leu Lys Ala Leu Glu Val Thr Glu Gln Arg Gln Lys 2090 2095 2100 Thr Ala Glu Lys Lys Asn Tyr Leu Leu Glu Glu Lys Ile Ala Ser 2105 2110 2115 Leu Ser Asn Ile Val Arg Asn Leu Thr Pro Ala Pro Leu Thr Ser 2120 2125 2130 Thr Pro Pro Leu Arg Ser 2135 7 3768 DNA Homo sapiens CDS (77)..(3766) 7 gccttggatt ttcaggtttt catcctgata cttgtttact tttctggggc agaaaagctt 60 gcactaattg ctctcc atg gtg gct aat ttt ttc aag agc ttg att tta cct 112 Met Val Ala Asn Phe Phe Lys Ser Leu Ile Leu Pro 1 5 10 tac att cat aag ctt tgc aaa gga atg ttt aca aag aaa ttg gga aat 160 Tyr Ile His Lys Leu Cys Lys Gly Met Phe Thr Lys Lys Leu Gly Asn 15 20 25 aca aac aaa aac aaa gag tat cgt cag cag aaa aag gat caa gac ttc 208 Thr Asn Lys Asn Lys Glu Tyr Arg Gln Gln Lys Lys Asp Gln Asp Phe 30 35 40 ccc act gct ggc cag acc aaa tcc ccc aaa ttt tct tac act ttt aaa 256 Pro Thr Ala Gly Gln Thr Lys Ser Pro Lys Phe Ser Tyr Thr Phe Lys 45 50 55 60 agc act gta aag aag att gca aag tgt tca tcc act cac aac tta tcc 304 Ser Thr Val Lys Lys Ile Ala Lys Cys Ser Ser Thr His Asn Leu Ser 65 70 75 act gag gaa gac gag gcc agt aaa gag ttt tcc ctc tca cca aca ttc 352 Thr Glu Glu Asp Glu Ala Ser Lys Glu Phe Ser Leu Ser Pro Thr Phe 80 85 90 agt tac cga gta gct att gcc aat ggc cta caa aag aat gct aaa gta 400 Ser Tyr Arg Val Ala Ile Ala Asn Gly Leu Gln Lys Asn Ala Lys Val 95 100 105 acc acc agt gat aat gag gat ctg ctt caa gag ctc tct tca atc gag 448 Thr Thr Ser Asp Asn Glu Asp Leu Leu Gln Glu Leu Ser Ser Ile Glu 110 115 120 agt tcc tac tca gaa tca tta aat gaa cta agg agt agc aca gaa aac 496 Ser Ser Tyr Ser Glu Ser Leu Asn Glu Leu Arg Ser Ser Thr Glu Asn 125 130 135 140 cag gca caa tca aca cac aca atg cca gtt aga cgc aac aga aag agt 544 Gln Ala Gln Ser Thr His Thr Met Pro Val Arg Arg Asn Arg Lys Ser 145 150 155 tca agc agc ctt gca ccc tct gag ggc agc tct gac ggg gag cgt act 592 Ser Ser Ser Leu Ala Pro Ser Glu Gly Ser Ser Asp Gly Glu Arg Thr 160 165 170 cta cat ggc tta aaa ctg gga gct tta cga aaa ctg aga aaa tgg aaa 640 Leu His Gly Leu Lys Leu Gly Ala Leu Arg Lys Leu Arg Lys Trp Lys 175 180 185 aag agt caa gaa tgt gtc tcc tca gac tca gag tta agc acc atg aaa 688 Lys Ser Gln Glu Cys Val Ser Ser Asp Ser Glu Leu Ser Thr Met Lys 190 195 200 aaa tcc tgg gga ata aga agt aag tct ttg gac aga act gtc cga aac 736 Lys Ser Trp Gly Ile Arg Ser Lys Ser Leu Asp Arg Thr Val Arg Asn 205 210 215 220 cca aag aca aat gcc ctg gag cca ggg ttc agt tcc tct ggc tgc att 784 Pro Lys Thr Asn Ala Leu Glu Pro Gly Phe Ser Ser Ser Gly Cys Ile 225 230 235 agc caa aca cat gat gtc atg gaa atg atc ttt aag gaa ctt cag gga 832 Ser Gln Thr His Asp Val Met Glu Met Ile Phe Lys Glu Leu Gln Gly 240 245 250 ata agt cag att gaa aca gaa ctt tct gaa cta cga ggg cac gtc aat 880 Ile Ser Gln Ile Glu Thr Glu Leu Ser Glu Leu Arg Gly His Val Asn 255 260 265 gct ctc aag cac tcc atc gat gag atc tcc agc agt gtg gag gtt gta 928 Ala Leu Lys His Ser Ile Asp Glu Ile Ser Ser Ser Val Glu Val Val 270 275 280 caa agt gaa att gag cag ttg cgc aca ggg ttt gtc cag tct cgg agg 976 Gln Ser Glu Ile Glu Gln Leu Arg Thr Gly Phe Val Gln Ser Arg Arg 285 290 295 300 gaa act aga gac atc cat gat tat att aag cac tta ggt cat atg ggt 1024 Glu Thr Arg Asp Ile His Asp Tyr Ile Lys His Leu Gly His Met Gly 305 310 315 agc aag gca agc ctg aga ttt tta aat gtg act gaa gaa aga ttt gaa 1072 Ser Lys Ala Ser Leu Arg Phe Leu Asn Val Thr Glu Glu Arg Phe Glu 320 325 330 tat gtt gaa agc gtg gtg tac caa att cta ata gat aaa atg ggt ttt 1120 Tyr Val Glu Ser Val Val Tyr Gln Ile Leu Ile Asp Lys Met Gly Phe 335 340 345 tca gat gca cca aat gct att aaa att gaa ttt gct cag agg ata gga 1168 Ser Asp Ala Pro Asn Ala Ile Lys Ile Glu Phe Ala Gln Arg Ile Gly 350 355 360 cac cag aga gac tgc cca aat gca aag cct cga ccc ata ctt gtg tac 1216 His Gln Arg Asp Cys Pro Asn Ala Lys Pro Arg Pro Ile Leu Val Tyr 365 370 375 380 ttt gaa acc cct caa caa agg gat tct gtc tta aaa aag tca tat aaa 1264 Phe Glu Thr Pro Gln Gln Arg Asp Ser Val Leu Lys Lys Ser Tyr Lys 385 390 395 ctc aaa gga aca ggc att gga atc tca aca gat att cta act cat gac 1312 Leu Lys Gly Thr Gly Ile Gly Ile Ser Thr Asp Ile Leu Thr His Asp 400 405 410 atc aga gaa aga aaa gag aaa ggg ata cca tcc tcc cag aca tat gag 1360 Ile Arg Glu Arg Lys Glu Lys Gly Ile Pro Ser Ser Gln Thr Tyr Glu 415 420 425 agc atg gct ata aag ttg tct act cca gag cca aaa atc aag aag aac 1408 Ser Met Ala Ile Lys Leu Ser Thr Pro Glu Pro Lys Ile Lys Lys Asn 430 435 440 aat tgg cag tca cct gat gac agt gat gaa gat ctt gaa tct gac ctc 1456 Asn Trp Gln Ser Pro Asp Asp Ser Asp Glu Asp Leu Glu Ser Asp Leu 445 450 455 460 aat aga aac agt tac gct gtg ctt tcc aag tca gag ctt cta aca aag 1504 Asn Arg Asn Ser Tyr Ala Val Leu Ser Lys Ser Glu Leu Leu Thr Lys 465 470 475 gga agt act tcc aag cca agc tca aaa tca cac agt gct aga tcc aag 1552 Gly Ser Thr Ser Lys Pro Ser Ser Lys Ser His Ser Ala Arg Ser Lys 480 485 490 aat aaa act gct aat agc agc aga att tca aat aaa tca gat tat gat 1600 Asn Lys Thr Ala Asn Ser Ser Arg Ile Ser Asn Lys Ser Asp Tyr Asp 495 500 505 aaa atc tcc tca cag ttg cca gaa tca gat atc ttg gaa aag caa acc 1648 Lys Ile Ser Ser Gln Leu Pro Glu Ser Asp Ile Leu Glu Lys Gln Thr 510 515 520 aca acc cat tat gca gat gca aca cct ctc tgg cac tca cag agt gat 1696 Thr Thr His Tyr Ala Asp Ala Thr Pro Leu Trp His Ser Gln Ser Asp 525 530 535 540 ttt ttc act gct aaa ctt agt cgt tct gaa tca gat ttt tcc aaa ttg 1744 Phe Phe Thr Ala Lys Leu Ser Arg Ser Glu Ser Asp Phe Ser Lys Leu 545 550 555 tgt cag tct tac tca gaa gat ttt tca gaa aat cag ttt ttc act aga 1792 Cys Gln Ser Tyr Ser Glu Asp Phe Ser Glu Asn Gln Phe Phe Thr Arg 560 565 570 act aat gga agc tct ctc ctg tca tct tcg gac cgg gag cta tgg cag 1840 Thr Asn Gly Ser Ser Leu Leu Ser Ser Ser Asp Arg Glu Leu Trp Gln 575 580 585 agg aaa cag gaa gga aca gcg acc ctg tat gac agt ccc aag gac cag 1888 Arg Lys Gln Glu Gly Thr Ala Thr Leu Tyr Asp Ser Pro Lys Asp Gln 590 595 600 cat ttg aat gga agt gtt cag ggt atc caa ggg cag act gaa act gaa 1936 His Leu Asn Gly Ser Val Gln Gly Ile Gln Gly Gln Thr Glu Thr Glu 605 610 615 620 aac aca gaa act gtg gat agt gga atg agt aat ggc atg gtg tgt gca 1984 Asn Thr Glu Thr Val Asp Ser Gly Met Ser Asn Gly Met Val Cys Ala 625 630 635 tct gga gac cgg agt cat tac agt gat tct cag ctc tct tta cat gag 2032 Ser Gly Asp Arg Ser His Tyr Ser Asp Ser Gln Leu Ser Leu His Glu 640 645 650 gat ctt tct cca tgg aag gaa tgg aat caa gga gct gat tta ggc ttg 2080 Asp Leu Ser Pro Trp Lys Glu Trp Asn Gln Gly Ala Asp Leu Gly Leu 655 660 665 gat tca tcc acc cag gaa ggt ttt gat tat gaa aca aac agt ctt ttt 2128 Asp Ser Ser Thr Gln Glu Gly Phe Asp Tyr Glu Thr Asn Ser Leu Phe 670 675 680 gac caa cag ctt gat gtt tac aat aaa gac cta gaa tac ttg gga aag 2176 Asp Gln Gln Leu Asp Val Tyr Asn Lys Asp Leu Glu Tyr Leu Gly Lys 685 690 695 700 tgc cac agt gat ctt caa gat gac tca gag agc tac gac tta act caa 2224 Cys His Ser Asp Leu Gln Asp Asp Ser Glu Ser Tyr Asp Leu Thr Gln 705 710 715 gat gac aat tct tct cca tgc cct ggc ttg gat aat gaa cca caa ggc 2272 Asp Asp Asn Ser Ser Pro Cys Pro Gly Leu Asp Asn Glu Pro Gln Gly 720 725 730 cag tgg gtt ggc caa tat gat tct tat cag gga gct aat tct aat gag 2320 Gln Trp Val Gly Gln Tyr Asp Ser Tyr Gln Gly Ala Asn Ser Asn Glu 735 740 745 cta tac caa aat caa aac cag ttg tcc atg atg tat cga agt caa agt 2368 Leu Tyr Gln Asn Gln Asn Gln Leu Ser Met Met Tyr Arg Ser Gln Ser 750 755 760 gaa ttg caa agt gat gat tca gag gat gcc cca ccc aaa tca tgg cat 2416 Glu Leu Gln Ser Asp Asp Ser Glu Asp Ala Pro Pro Lys Ser Trp His 765 770 775 780 agt cga tta agc att gac ctt tct gat aag act ttc agc ttc cca aaa 2464 Ser Arg Leu Ser Ile Asp Leu Ser Asp Lys Thr Phe Ser Phe Pro Lys 785 790 795 ttt gga tct aca ctg cag agg gct aaa tca gcc ttg gaa gta gta tgg 2512 Phe Gly Ser Thr Leu Gln Arg Ala Lys Ser Ala Leu Glu Val Val Trp 800 805 810 aac aaa agc aca cag agt ctg agt ggg tat gag gac agt ggc tct tca 2560 Asn Lys Ser Thr Gln Ser Leu Ser Gly Tyr Glu Asp Ser Gly Ser Ser 815 820 825 tta atg ggg aga ttt cgg aca tta tct caa tca act gca aat gag tca 2608 Leu Met Gly Arg Phe Arg Thr Leu Ser Gln Ser Thr Ala Asn Glu Ser 830 835 840 agt acc aca ctt gac tct gat gtc tac acg gag ccc tat tac tat aaa 2656 Ser Thr Thr Leu Asp Ser Asp Val Tyr Thr Glu Pro Tyr Tyr Tyr Lys 845 850 855 860 gca gag gat gag gaa gat tat act gaa cca gtg gct gac aat gaa aca 2704 Ala Glu Asp Glu Glu Asp Tyr Thr Glu Pro Val Ala Asp Asn Glu Thr 865 870 875 gat tat gtt gaa gtc atg gaa caa gtc ctt gct aaa cta gaa aac agg 2752 Asp Tyr Val Glu Val Met Glu Gln Val Leu Ala Lys Leu Glu Asn Arg 880 885 890 act agt att act gaa aca gat gaa caa atg caa gca tat gat cac ctt 2800 Thr Ser Ile Thr Glu Thr Asp Glu Gln Met Gln Ala Tyr Asp His Leu 895 900 905 tca tat gaa aca cct tat gaa acc cca caa gat gag ggt tat gat ggt 2848 Ser Tyr Glu Thr Pro Tyr Glu Thr Pro Gln Asp Glu Gly Tyr Asp Gly 910 915 920 cca gca gat gat atg gtt agt gaa gag ggg tta gaa ccc tta aat gaa 2896 Pro Ala Asp Asp Met Val Ser Glu Glu Gly Leu Glu Pro Leu Asn Glu 925 930 935 940 aca tca gct gag atg gaa ata aga gaa gat gaa aac caa aac att cct 2944 Thr Ser Ala Glu Met Glu Ile Arg Glu Asp Glu Asn Gln Asn Ile Pro 945 950 955 gaa cag cca gtg gag atc aca aag cca aag aga att cgt cct tct ttc 2992 Glu Gln Pro Val Glu Ile Thr Lys Pro Lys Arg Ile Arg Pro Ser Phe 960 965 970 aaa gaa gca gct tta agg gcc tat aaa aag caa atg gca gag ttg gaa 3040 Lys Glu Ala Ala Leu Arg Ala Tyr Lys Lys Gln Met Ala Glu Leu Glu 975 980 985 gag aag atc ttg gct gga gat agc agt tct gtg gat gaa aag gct cga 3088 Glu Lys Ile Leu Ala Gly Asp Ser Ser Ser Val Asp Glu Lys Ala Arg 990 995 1000 ata gta agt ggc aat gat ttg gat gct tcc aaa ttt tct gca ctc 3133 Ile Val Ser Gly Asn Asp Leu Asp Ala Ser Lys Phe Ser Ala Leu 1005 1010 1015 cag gtg tgt ggt ggg gct gga ggt gga ctt tat ggt att gac agc 3178 Gln Val Cys Gly Gly Ala Gly Gly Gly Leu Tyr Gly Ile Asp Ser 1020 1025 1030 atg ccg gat ctt cgc aga aaa aaa act ttg cct att gtc cga gat 3223 Met Pro Asp Leu Arg Arg Lys Lys Thr Leu Pro Ile Val Arg Asp 1035 1040 1045 gtg gcc atg acc ctg gct gcc cgg aaa tct gga ctc tcc ctg gct 3268 Val Ala Met Thr Leu Ala Ala Arg Lys Ser Gly Leu Ser Leu Ala 1050 1055 1060 atg gtg att agg aca tcc cta aat aat gag gaa ctg aaa atg cac 3313 Met Val Ile Arg Thr Ser Leu Asn Asn Glu Glu Leu Lys Met His 1065 1070 1075 gtc ttc aag aag acc ttg cag gca ctg atc tac cct atg tct tct 3358 Val Phe Lys Lys Thr Leu Gln Ala Leu Ile Tyr Pro Met Ser Ser 1080 1085 1090 acc atc cca cac aat ttt gag gtc tgg acg gct acc aca ccc acc 3403 Thr Ile Pro His Asn Phe Glu Val Trp Thr Ala Thr Thr Pro Thr 1095 1100 1105 tac tgt tat gag tgt gaa ggg ctc ctg tgg ggc att gca agg caa 3448 Tyr Cys Tyr Glu Cys Glu Gly Leu Leu Trp Gly Ile Ala Arg Gln 1110 1115 1120 ggc atg aag tgt ctg gag tgt gga gtg aaa tgc cac gaa aag tgt 3493 Gly Met Lys Cys Leu Glu Cys Gly Val Lys Cys His Glu Lys Cys 1125 1130 1135 cag gac ctg cta aac gct gac tgc ttg cag aga gca gca gaa aag 3538 Gln Asp Leu Leu Asn Ala Asp Cys Leu Gln Arg Ala Ala Glu Lys 1140 1145 1150 agt tct aaa cat ggt gcc gaa gac aag act cag acc att att aca 3583 Ser Ser Lys His Gly Ala Glu Asp Lys Thr Gln Thr Ile Ile Thr 1155 1160 1165 gca atg aaa gaa aga atg aag atc agg gag aaa aac cgg cca gaa 3628 Ala Met Lys Glu Arg Met Lys Ile Arg Glu Lys Asn Arg Pro Glu 1170 1175 1180 gta ttt gaa gta atc cag gaa atg ttt cag att tct aaa gaa gat 3673 Val Phe Glu Val Ile Gln Glu Met Phe Gln Ile Ser Lys Glu Asp 1185 1190 1195 ttt gtg cag ttt aca aag gcg gcc aaa cag agt gta ctg gat ggg 3718 Phe Val Gln Phe Thr Lys Ala Ala Lys Gln Ser Val Leu Asp Gly 1200 1205 1210 aca tct aag tgg tct gca aaa ata acc atc aca gtg gtt tct gca 3763 Thr Ser Lys Trp Ser Ala Lys Ile Thr Ile Thr Val Val Ser Ala 1215 1220 1225 caa gg 3768 Gln 1230 8 1230 PRT Homo sapiens 8 Met Val Ala Asn Phe Phe Lys Ser Leu Ile Leu Pro Tyr Ile His Lys 1 5 10 15 Leu Cys Lys Gly Met Phe Thr Lys Lys Leu Gly Asn Thr Asn Lys Asn 20 25 30 Lys Glu Tyr Arg Gln Gln Lys Lys Asp Gln Asp Phe Pro Thr Ala Gly 35 40 45 Gln Thr Lys Ser Pro Lys Phe Ser Tyr Thr Phe Lys Ser Thr Val Lys 50 55 60 Lys Ile Ala Lys Cys Ser Ser Thr His Asn Leu Ser Thr Glu Glu Asp 65 70 75 80 Glu Ala Ser Lys Glu Phe Ser Leu Ser Pro Thr Phe Ser Tyr Arg Val 85 90 95 Ala Ile Ala Asn Gly Leu Gln Lys Asn Ala Lys Val Thr Thr Ser Asp 100 105 110 Asn Glu Asp Leu Leu Gln Glu Leu Ser Ser Ile Glu Ser Ser Tyr Ser 115 120 125 Glu Ser Leu Asn Glu Leu Arg Ser Ser Thr Glu Asn Gln Ala Gln Ser 130 135 140 Thr His Thr Met Pro Val Arg Arg Asn Arg Lys Ser Ser Ser Ser Leu 145 150 155 160 Ala Pro Ser Glu Gly Ser Ser Asp Gly Glu Arg Thr Leu His Gly Leu 165 170 175 Lys Leu Gly Ala Leu Arg Lys Leu Arg Lys Trp Lys Lys Ser Gln Glu 180 185 190 Cys Val Ser Ser Asp Ser Glu Leu Ser Thr Met Lys Lys Ser Trp Gly 195 200 205 Ile Arg Ser Lys Ser Leu Asp Arg Thr Val Arg Asn Pro Lys Thr Asn 210 215 220 Ala Leu Glu Pro Gly Phe Ser Ser Ser Gly Cys Ile Ser Gln Thr His 225 230 235 240 Asp Val Met Glu Met Ile Phe Lys Glu Leu Gln Gly Ile Ser Gln Ile 245 250 255 Glu Thr Glu Leu Ser Glu Leu Arg Gly His Val Asn Ala Leu Lys His 260 265 270 Ser Ile Asp Glu Ile Ser Ser Ser Val Glu Val Val Gln Ser Glu Ile 275 280 285 Glu Gln Leu Arg Thr Gly Phe Val Gln Ser Arg Arg Glu Thr Arg Asp 290 295 300 Ile His Asp Tyr Ile Lys His Leu Gly His Met Gly Ser Lys Ala Ser 305 310 315 320 Leu Arg Phe Leu Asn Val Thr Glu Glu Arg Phe Glu Tyr Val Glu Ser 325 330 335 Val Val Tyr Gln Ile Leu Ile Asp Lys Met Gly Phe Ser Asp Ala Pro 340 345 350 Asn Ala Ile Lys Ile Glu Phe Ala Gln Arg Ile Gly His Gln Arg Asp 355 360 365 Cys Pro Asn Ala Lys Pro Arg Pro Ile Leu Val Tyr Phe Glu Thr Pro 370 375 380 Gln Gln Arg Asp Ser Val Leu Lys Lys Ser Tyr Lys Leu Lys Gly Thr 385 390 395 400 Gly Ile Gly Ile Ser Thr Asp Ile Leu Thr His Asp Ile Arg Glu Arg 405 410 415 Lys Glu Lys Gly Ile Pro Ser Ser Gln Thr Tyr Glu Ser Met Ala Ile 420 425 430 Lys Leu Ser Thr Pro Glu Pro Lys Ile Lys Lys Asn Asn Trp Gln Ser 435 440 445 Pro Asp Asp Ser Asp Glu Asp Leu Glu Ser Asp Leu Asn Arg Asn Ser 450 455 460 Tyr Ala Val Leu Ser Lys Ser Glu Leu Leu Thr Lys Gly Ser Thr Ser 465 470 475 480 Lys Pro Ser Ser Lys Ser His Ser Ala Arg Ser Lys Asn Lys Thr Ala 485 490 495 Asn Ser Ser Arg Ile Ser Asn Lys Ser Asp Tyr Asp Lys Ile Ser Ser 500 505 510 Gln Leu Pro Glu Ser Asp Ile Leu Glu Lys Gln Thr Thr Thr His Tyr 515 520 525 Ala Asp Ala Thr Pro Leu Trp His Ser Gln Ser Asp Phe Phe Thr Ala 530 535 540 Lys Leu Ser Arg Ser Glu Ser Asp Phe Ser Lys Leu Cys Gln Ser Tyr 545 550 555 560 Ser Glu Asp Phe Ser Glu Asn Gln Phe Phe Thr Arg Thr Asn Gly Ser 565 570 575 Ser Leu Leu Ser Ser Ser Asp Arg Glu Leu Trp Gln Arg Lys Gln Glu 580 585 590 Gly Thr Ala Thr Leu Tyr Asp Ser Pro Lys Asp Gln His Leu Asn Gly 595 600 605 Ser Val Gln Gly Ile Gln Gly Gln Thr Glu Thr Glu Asn Thr Glu Thr 610 615 620 Val Asp Ser Gly Met Ser Asn Gly Met Val Cys Ala Ser Gly Asp Arg 625 630 635 640 Ser His Tyr Ser Asp Ser Gln Leu Ser Leu His Glu Asp Leu Ser Pro 645 650 655 Trp Lys Glu Trp Asn Gln Gly Ala Asp Leu Gly Leu Asp Ser Ser Thr 660 665 670 Gln Glu Gly Phe Asp Tyr Glu Thr Asn Ser Leu Phe Asp Gln Gln Leu 675 680 685 Asp Val Tyr Asn Lys Asp Leu Glu Tyr Leu Gly Lys Cys His Ser Asp 690 695 700 Leu Gln Asp Asp Ser Glu Ser Tyr Asp Leu Thr Gln Asp Asp Asn Ser 705 710 715 720 Ser Pro Cys Pro Gly Leu Asp Asn Glu Pro Gln Gly Gln Trp Val Gly 725 730 735 Gln Tyr Asp Ser Tyr Gln Gly Ala Asn Ser Asn Glu Leu Tyr Gln Asn 740 745 750 Gln Asn Gln Leu Ser Met Met Tyr Arg Ser Gln Ser Glu Leu Gln Ser 755 760 765 Asp Asp Ser Glu Asp Ala Pro Pro Lys Ser Trp His Ser Arg Leu Ser 770 775 780 Ile Asp Leu Ser Asp Lys Thr Phe Ser Phe Pro Lys Phe Gly Ser Thr 785 790 795 800 Leu Gln Arg Ala Lys Ser Ala Leu Glu Val Val Trp Asn Lys Ser Thr 805 810 815 Gln Ser Leu Ser Gly Tyr Glu Asp Ser Gly Ser Ser Leu Met Gly Arg 820 825 830 Phe Arg Thr Leu Ser Gln Ser Thr Ala Asn Glu Ser Ser Thr Thr Leu 835 840 845 Asp Ser Asp Val Tyr Thr Glu Pro Tyr Tyr Tyr Lys Ala Glu Asp Glu 850 855 860 Glu Asp Tyr Thr Glu Pro Val Ala Asp Asn Glu Thr Asp Tyr Val Glu 865 870 875 880 Val Met Glu Gln Val Leu Ala Lys Leu Glu Asn Arg Thr Ser Ile Thr 885 890 895 Glu Thr Asp Glu Gln Met Gln Ala Tyr Asp His Leu Ser Tyr Glu Thr 900 905 910 Pro Tyr Glu Thr Pro Gln Asp Glu Gly Tyr Asp Gly Pro Ala Asp Asp 915 920 925 Met Val Ser Glu Glu Gly Leu Glu Pro Leu Asn Glu Thr Ser Ala Glu 930 935 940 Met Glu Ile Arg Glu Asp Glu Asn Gln Asn Ile Pro Glu Gln Pro Val 945 950 955 960 Glu Ile Thr Lys Pro Lys Arg Ile Arg Pro Ser Phe Lys Glu Ala Ala 965 970 975 Leu Arg Ala Tyr Lys Lys Gln Met Ala Glu Leu Glu Glu Lys Ile Leu 980 985 990 Ala Gly Asp Ser Ser Ser Val Asp Glu Lys Ala Arg Ile Val Ser Gly 995 1000 1005 Asn Asp Leu Asp Ala Ser Lys Phe Ser Ala Leu Gln Val Cys Gly 1010 1015 1020 Gly Ala Gly Gly Gly Leu Tyr Gly Ile Asp Ser Met Pro Asp Leu 1025 1030 1035 Arg Arg Lys Lys Thr Leu Pro Ile Val Arg Asp Val Ala Met Thr 1040 1045 1050 Leu Ala Ala Arg Lys Ser Gly Leu Ser Leu Ala Met Val Ile Arg 1055 1060 1065 Thr Ser Leu Asn Asn Glu Glu Leu Lys Met His Val Phe Lys Lys 1070 1075 1080 Thr Leu Gln Ala Leu Ile Tyr Pro Met Ser Ser Thr Ile Pro His 1085 1090 1095 Asn Phe Glu Val Trp Thr Ala Thr Thr Pro Thr Tyr Cys Tyr Glu 1100 1105 1110 Cys Glu Gly Leu Leu Trp Gly Ile Ala Arg Gln Gly Met Lys Cys 1115 1120 1125 Leu Glu Cys Gly Val Lys Cys His Glu Lys Cys Gln Asp Leu Leu 1130 1135 1140 Asn Ala Asp Cys Leu Gln Arg Ala Ala Glu Lys Ser Ser Lys His 1145 1150 1155 Gly Ala Glu Asp Lys Thr Gln Thr Ile Ile Thr Ala Met Lys Glu 1160 1165 1170 Arg Met Lys Ile Arg Glu Lys Asn Arg Pro Glu Val Phe Glu Val 1175 1180 1185 Ile Gln Glu Met Phe Gln Ile Ser Lys Glu Asp Phe Val Gln Phe 1190 1195 1200 Thr Lys Ala Ala Lys Gln Ser Val Leu Asp Gly Thr Ser Lys Trp 1205 1210 1215 Ser Ala Lys Ile Thr Ile Thr Val Val Ser Ala Gln 1220 1225 1230 

What is claimed is:
 1. An isolated protein complex comprising two proteins, the protein complex selected from the group consisting of (a) a complex set forth in Table 1; (b) a complex set forth in Table 2; (c) a complex set forth in Table 3; (d) a complex set forth in Table 4; (d) a complex set forth in Table 5; (d) a complex set forth in Table 6; (d) a complex set forth in Table 7; (d) a complex set forth in Table 8; (d) a complex set forth in Table 9; (d) a complex set forth in Table 10; (d) a complex set forth in Table 11; (d) a complex set forth in Table 12; (d) a complex set forth in Table 13; (d) a complex set forth in Table 14; (d) a complex set forth in Table 15; (d) a complex set forth in Table 16; (d) a complex set forth in Table 17; (d) a complex set forth in Table 18; (d) a complex set forth in Table 19; (d) a complex set forth in Table 20; (d) a complex set forth in Table 21; (d) a complex set forth in Table 22; (d) a complex set forth in Table 23; (d) a complex set forth in Table 24; (d) a complex set forth in Table 25; (d) a complex set forth in Table 26; (d) a complex set forth in Table 27; (d) a complex set forth in Table 28; (d) a complex set forth in Table 29; (d) a complex set forth in Table 30; (d) a complex set forth in Table 31; and (d) a complex set forth in Table
 32. 2. The protein complex of claim 1, wherein said protein complex comprises complete proteins.
 3. The protein complex of claim 1, wherein said protein complex comprises a fragment of one protein and a complete protein of anther protein.
 4. The protein complex of claim 1, wherein said protein complex comprises fragments of proteins.
 5. An isolated antibody selectively immunoreactive with a protein complex of claim
 1. 6. The antibody of claim 5, wherein said antibody is a monoclonal antibody.
 7. A method for diagnosing a physiological disorder in an animal, which comprises assaying for: (a) whether a protein complex set forth in any one of Tables 1-31 is present in a tissue extract; (b) the ability of proteins to form a protein complex set forth in any one of Tables 1-31; and (c) a mutation in a gene encoding a protein of a protein complex set forth in any one of Tables 1-31.
 8. The method of claim 7, wherein said animal is a human.
 9. The method of claim 7, wherein the diagnosis is for a predisposition to said physiological disorder.
 10. The method of claim 7, wherein the diagnosis is for the existence of said physiological disorder.
 11. The method of claim 7, wherein said assay comprises a yeast two-hybrid assay.
 12. The method of claim 7, wherein said assay comprises measuring in vitro a complex formed by combining the proteins of the protein complex, said proteins isolated from said animal.
 13. The method of claim 12, wherein said complex is measured by binding with an antibody specific for said complex.
 14. The method of claim 7, wherein said assay comprises mixing an antibody specific for said protein complex with a tissue extract from said animal and measuring the binding of said antibody.
 15. A method for determining whether a mutation in a gene encoding one of the proteins of a protein complex set forth in any one of Tables 1-31 is useful for diagnosing a physiological disorder, which comprises assaying for the ability of said protein with said mutation to form a complex with the other protein of said protein complex, wherein an inability to form said complex is indicative of said mutation being useful for diagnosing a physiological disorder.
 16. The method of claim 15, wherein said gene is an animal gene.
 17. The method of claim 16, wherein said animal is a human.
 18. The method of claim 15, wherein the diagnosis is for a predisposition to a physiological disorder.
 19. The method of claim 15, wherein the diagnosis is for the existence of a physiological disorder.
 20. The method of claim 15, wherein said assay comprises a yeast two-hybrid assay.
 21. The method of claim 15, wherein said assay comprises measuring in vitro a complex formed by combining the proteins of the protein complex, said proteins isolated from an animal.
 22. The method of claim 21, wherein said animal is a human.
 23. The method of claim 21, wherein said complex is measured by binding with an antibody specific for said complex.
 24. A method for screening for drug candidates capable of modulating the interaction of the proteins of a protein complex set forth in any one of Tables 1-31, which comprises: (a) combining the proteins of said protein complex in the presence of a drug to form a first complex; (b) combining the proteins in the absence of said drug to form a second complex; (c) measuring the amount of said first complex and said second complex; and (d) comparing the amount of said first complex with the amount of said second complex, wherein if the amount of said first complex is greater than, or less than the amount of said second complex, then the drug is a drug candidate for modulating the interaction of the proteins of said protein complex.
 25. The method of claim 24, wherein said screening is an in vitro screening.
 26. The method of claim 24, wherein said complex is measured by binding with an antibody specific for said protein complexes.
 27. The method of claim 24, wherein if the amount of said first complex is greater than the amount of said second complex, then said drug is a drug candidate for promoting the interaction of said proteins.
 28. The method of claim 24, wherein if the amount of said first complex is less than the amount of said second complex, then said drug is a drug candidate for inhibiting the interaction of said proteins.
 29. A non-human animal model for a physiological disorder wherein the genome of said animal or an ancestor thereof has been modified such that the formation of a protein complex set forth in any one of Tables 1-31 has been altered.
 30. The non-human animal model of claim 29, wherein the formation of said protein complex has been altered as a result of: (a) over-expression of at least one of the proteins of said protein complex; (b) replacement of a gene for at least one of the proteins of said protein complex with a gene from a second animal and expression of said protein; (c) expression of a mutant form of at least one of the proteins of said protein complex; (d) a lack of expression of at least one of the proteins of said protein complex; or (e) reduced expression of at least one of the proteins of said protein complex.
 31. A cell line obtained from the animal model of claim
 29. 32. A non-human animal model for a physiological disorder, wherein the biological activity of a protein complex set forth in any one of Tables 1-31 has been altered.
 33. The non-human animal model of claim 32, wherein said biological activity has been altered as a result of: (a) disrupting the formation of said complex; or (b) disrupting the action of said complex.
 34. The non-human animal model of claim 32, wherein the formation of said complex is disrupted by binding an antibody to at least one of the proteins which form said protein complex.
 35. The non-human animal model of claim 32, wherein the action of said complex is disrupted by binding an antibody to said complex.
 36. The non-human animal model of claim 32, wherein the formation of said complex is disrupted by binding a small molecule to at least one of the proteins which form said protein complex.
 37. The non-human animal model of claim 32, wherein the action of said complex is disrupted by binding a small molecule to said complex.
 38. A cell in which the genome of cells of said cell line has been modified to produce at least one protein complex set forth in any one of Tables 1-31.
 39. A cell line in which the genome of the cells of said cell line has been modified to eliminate at least one protein of a protein complex set forth in any one of Tables 1-31.
 40. A method of screening for drug candidates useful in treating a physiological disorder which comprises the steps of: (a) measuring the activity of a protein selected from the proteins set forth in Tables 1-31 in the presence of a drug, (b) measuring the activity of said protein in the absence of said drug, and (c) comparing the activity measured in steps (1) and (2), wherein if there is a difference in activity, then said drug is a drug candidate for treating said physiological disorder.
 41. An isolated nucleic acid comprising a nucleic acid coding for a protein comprising an amino acid sequence selected from the group of amino acid sequences set forth in SEQ ID NOs:4, 6 and 8 and amino acid sequences having at least 95% identity to the amino acid sequences set forth in SEQ ID NOs:4, 6 and
 8. 42. The nucleic acid of claim 41 wherein the nucleic acid comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NOs:3, 5 and 7, nucleotide sequences having at least 95% identity to the nucleotide sequences set forth in SEQ ID NOs:3, 5 and 7 and their complements.
 43. A substantially pure protein comprising an amino acid sequence selected from the group of amino acid sequences set forth in SEQ ID NOs:4, 6 and 8 and amino acid sequences having al least 95% identity to the amino acid sequences set forth in SEQ ID NOs:4, 6 and
 8. 44. An antibody specific for the protein of claim
 43. 45. The antibody of claim 44 which is a monoclonal antibody. 